Prevention and treatment of amyloidogenic diseases

ABSTRACT

Disclosed are pharmaceutical compositions and methods for preventing or treating a number of amyloid diseases, including Alzheimer&#39;s disease, prion diseases, familial amyloid neuropathies and the like. The pharmaceutical compositions include immunologically reactive amounts of amyloid fibril components, particularly fibril-forming peptides or proteins. Also disclosed are therapeutic compositions and methods which use immune reagents that react with such fibril components.

This application claims priority to U.S. Provisional Application60/137,010, filed Jun. 1, 1999, which is hereby incorporated herein inits entirety.

FIELD OF THE INVENTION

The invention relates to compositions and methods of treatment ofamyloid-related conditions in humans and other mammalian vertebrates.

BACKGROUND OF THE INVENTION

Amyloidosis is a general term that describes a number of diseasescharacterized by extracellular deposition of protein fibrils, which formnumerous “amyloid deposits,” which may occur in localized sites orsystemically. The fibrillar composition of these deposits is anidentifying characteristic for the various forms of amyloid disease. Forexample, intracerebral and cerebrovascular deposits composed primarilyof fibrils of beta amyloid peptide (β-AP) are characteristic ofAlzheimer's disease (both familial and sporadic forms), islet amyloidprotein peptide (IAPP; amylin) is characteristic of the fibrils inpancreatic islet cell amyloid deposits associated with type II diabetes,and β2-microglobulin is a major component of amyloid deposits which formas a consequence of long term hemodialysis treatment. More recently,prion-associated diseases, such as Creutzfeld-Jacob disease, have alsobeen recognized as amyloid diseases.

The various forms of disease have been divided into classes, mostly onthe basis of whether or not the amyloidosis is associated with anunderlying systemic illness. Thus, certain disorders are considered tobe primary amyloidoses, in which there is no evidence for preexisting orcoexisting disease. In general, primary amyloidoses of the disease arecharacterized by the presence of “amyloid light chain-type” (AL-type)protein fibrils, so named for the homology of the N-terminal region ofthe AL fibrils to the variable fragment of immunoglobulin light chain(kappa or lambda).

Secondary or “reactive” amyloidosis is characterized by deposition of AAtype fibrils derived from serum amyloid A protein (ApoSSA). These formsof amyloidosis are characterized by an underlying chronic inflammatoryor infectious disease state (e.g., rheumatoid arthritis, osteomyelitis,tuberculosis, leprosy).

characterized by an underlying chronic inflammatory or infectiousdisease state (e.g., rheumatoid arthritis, osteomyelitis, tuberculosis,leprosy).

Heredofamilial amyloidoses may have associated neuropathic, renal, orcardiovascular deposits of the ATTR transthyretin type. Otherheredofamilial amyloidoses include other syndromes and may havedifferent amyloid components (e.g., familial Mediterranean fever whichis characterized by AA fibrils). Other forms of amyloidosis includelocal forms, characterized by focal, often tumor-like deposits thatoccur in isolated organs. Other amyloidoses are associated with aging,and are commonly characterized by plaque formation in the heart orbrain. Also common are amyloid deposits associated with long termhemodialysis. These and other forms of amyloid disease are summarized inTable 1. (Tan, S. Y. and Pepys, Histopathology 25:403-414, 1994;Harrison's Handbook of Internal Medicine, 13^(th) Ed., Isselbacher, K.J., et al, eds, McGraw-Hill, San Francisco, 1995).

TABLE 1 Classification of Amyloid Diseases Amyloid ProteinProtein/Peptide Precursor Protein Variants Clinical AA Serum Amyloid AReactive (secondary) Protein (ApoSSA) Amyloidosis: FamilialMediterranean fever Familial amyloid nephropathy with urticaria anddeafness (Muckle- Wells syndrome) AA Serum amyloid A Reactive systemicprotein amyloidosis associated (ApoSSA) with systemic inflammatorydiseases AL Monoclonal Ak, A, (e.g., Idiopathic (primary) immunoglobulinlight AkIII) Amyloidosis: myeloma or chains (kappa, lambda)macroglobulinemia- associated; systemic amyloidosis associated withimmunocyte dyscrasia; monoclonal gammopathy; occult dyscrasia; localnodular amyloidosis associated with chronic inflammatory diseases AH IgG(1(γ1)) Aγ1 Heavy chain amyloidosis associated with several immunocytedyscrasias ATTR Transthyretin (TTR) At least 30 Familial amyloid knownpoint polyneuropathy mutations (e.g., Met 30, Portuguese) ATTRTransthyretin (TTR) e.g., Met 111 Familial amyloid cardiomyopathy(Danish) ATTR Transthyretin (TTR) Wild-type TTR Systemic senile or Ile122 amyloidosis AapoAI ApoAI Arg 26 Familial amyloid polyneuropathy AgelGelsolin Asn 187 Familial amyloidosis (Finnish) Acys Cystatin C Gln 68Hereditary cerebral hemorrhage with amyloidosis (Icelandic) Aβ Amyloid βprotein Various: Gln Alzheimer's disease precursor (e.g. β-APP₆₉₅) 618,Down's syndrome Hereditary cerebral hemorrhage amyloidosis (Dutch)Sporadic cerebral amyloid angiopathy Inclusion body myositis AB₂M Beta₂microglobulin Associated with chronic hemodialysis Acal (Pro)calcitonin(Pro)calcitonin Medullary carcinoma of thyroid AANF Atrial natriureticfactor Focal Senile Amyloidoses: Isolated atrial amyloid Aβ β-amyloidprecursor Brain protein SVEP^(a) — Seminal vesicles AB₂M Beta₂microglobulin Prostate Keratin Primary localized cutaneous amyloid(macular, papular) PrP Prion precursor protein Scrapie protein SporadicCreutzfeldt-Jacob (33-35 kDa cellular 27-30 kDa Disease form) Kuru(transmissible spongiform encephalopathies, prion diseases) AIAPP Isletamyloid Islets of Langerhans polypeptide (IAPP) Diabetes type II,Insulinoma Peptide e.g., precalcitonin Exocrine amyloidosis, hormones,associated with fragments APUDomas ^(a)Seminal vesicle exocrine protein

Often, fibrils forming the bulk of an amyloid deposit are derived fromone or more primary precursor proteins or peptides, and are usuallyassociated with sulfated glycosaminoglycans. In addition, amyloiddeposits may include minor proteins and peptides of various types, alongwith other components, such as proteoglycans, gangliosides and othersugars, as described in more detail in the sections that follow.

Currently, there are no specific, amyloid-directed treatments for any ofthe amyloid diseases. Where there is an underlying or associated diseasestate, therapy is directed towards decreasing the production ofamyloidogenic protein by treating the underlying disease. This isexemplified by the treatment of tuberculosis with antibiotics, therebyreducing the mycobacterial load, resulting in a reduction ofinflammation and in associated reduction of SSA protein. In the case ofAL amyloid due to multiple myeloma, chemotherapy is administered topatients, causing a reduction in plasma cells and a lowering of myelomaimmunoglobulin levels. As these levels decline, the AL amyloid mayclear. Co-owned U.S. patent applications U.S. Ser. No. 09/201,430, filedNov. 30, 1998 and U.S. Ser. No. 09/322,289, filed May 28, 1999 revealthat amyloid plaque burden associated with Alzheimer's disease can begreatly reduced (and prevented) by administration of agents whichproduce or confer an immune response directed at β-amyloid peptide (Aβ)and fragments thereof. It is the discovery of the present invention thatinduction of an immune response to various amyloid plaque components iseffective in treating a broad range of amyloid diseases.

SUMMARY OF THE INVENTION

The present invention is directed to pharmaceutical compositions andmethods for treating a number of amyloid diseases. According to oneaspect, the invention includes pharmaceutical compositions that include,as an active ingredient, an agent that is effective to induce an immuneresponse against an amyloid component in a patient. Such compositionswill generally also include excipients and in preferred embodiments mayinclude adjuvants. In further preferred embodiments, the adjuvantsinclude, for example, aluminum hydroxide, aluminum phosphate, MPL™,QS-21 (Stimulon™) or incomplete Freund's adjuvant. According to arelated embodiment, such pharmaceutical compositions may include aplurality of agents effective to induce an immune response against morethan one amyloid component in the patient.

In a related embodiment, the agent is effective to produce an immuneresponse directed against a fibril peptide or protein amyloid component.Preferably, such a fibril peptide or protein is derived from a fibrilprecursor protein known to be associated with certain forms of amyloiddiseases, as described herein. Such precursor proteins include, but arenot limited to, Serum Amyloid A protein (ApoSSA), immunoglobulin lightchain, immunoglobulin heavy chain, ApoAI, transthyretin, lysozyme,fibrogen α chain, gelsolin, cystatin C, Amyloid β protein precursor(β-APP), Beta₂ microglobulin, prion precursor protein (PrP), atrialnatriuretic factor, keratin, islet amyloid polypeptide, a peptidehormone, and synuclein. Such precursors also include mutant proteins,protein fragments and proteolytic peptides of such precursors. In apreferred embodiment, the agent is effective to induce an immuneresponse directed against a neoepitope formed by a fibril protein orpeptide, with respect to a fibril precursor protein. That is, asdescribed in more detail herein, many fibril-forming peptides orproteins are fragments of such precursor proteins, such as those listedabove. When such fragments are formed, such as by proteolytic cleavage,epitopes may be revealed that are not present on the precursor and aretherefore not immunologically available to the immune system when thefragment is a part of the precursor protein. Agents directed to suchepitopes may be preferred therapeutic agents, since they may be lesslikely to induce an autoimmune response in the patient.

According to a related embodiment, pharmaceutical compositions of theinvention include agents directed to amyloid components, such as thoseselected from the group including, but not limited to the followingfibril peptides or proteins: AA, AL, ATTR, AApoA1, Alys, Agel, Acys, Aβ,AB₂M, AScr, Acal, AIAPP and synuclein-NAC fragment. The full names andcompositions of these peptides are described herein. Such peptides canbe made according to methods well known in the art, as described herein.

According to a further related embodiment, agents included in suchpharmaceutical compositions also include certain to sulfatedproteoglycans. In a related embodiment, the proteoglycan is a heparinsulfate glycosaminoglycan, preferably perlecan, dermatan sulfate,chondroitin-4-sulfate, or pentosan polysulfate.

According to another related aspect, the invention includes a method ofpreventing or treating a disorder characterized by amyloid deposition ina mammalian subject. In accordance with this aspect of the invention,the subject is given a dosage of an agent effective to produce an immuneresponse against an amyloid component characteristic of the amyloiddisorder from which the subject suffers. Essentially, the methodsinclude administering pharmaceutical compositions containing immunogenicamyloid components specific to the disorder, such as those describedabove. Such methods are further characterized by their effectiveness ininducing immunogenic responses in the subject. According to a preferredembodiment, the method is effective to produce an immunological responsethat is characterized by a serum titer of at least 1:1000 with respectto the amyloid component against which the immunogenic agent isdirected. In yet a further preferred embodiment, the serum titer is atleast 1:5000 with respect to the fibril component. According to arelated embodiment, the immune response is characterized by a serumamount of immunoreactivity corresponding to greater than about fourtimes higher than a serum level of immunoreactivity measured in apre-treatment control serum sample. This latter characterization isparticularly appropriate when serum immunoreactivity is measured byELISA techniques, but can apply to any relative or absolute measurementof serum immunoreactivity. According to a preferred embodiment, theimmunoreactivity is measured at a serum dilution of about 1:100.

According to a still further related aspect, the invention includes amethod of determining the prognosis of a patient undergoing treatmentfor an amyloid disorder. Here, patient serum amount of immunoreactivityagainst an amyloid component characteristic of the selected disorder ismeasured, and a patient serum amount of immunoreactivity of at leastfour times a baseline control level of serum immunoreactivity isindicative of a prognosis of improved status with respect to theparticular amyloid disorder. According to preferred embodiments, theamount of immunoreactivity against the selected amyloid componentpresent in the patient serum is characterized by a serum titer of atleast about 1:1000, or at least 1:5000, with respect to the amyloidcomponent.

According to a still related aspect, the invention also includesso-called “passive immunization” methods and pharmaceutical compositionsfor preventing or treating amyloid diseases. According to this aspect ofthe invention, patients are given an effective dosage of an antibodythat specifically binds to a selected amyloid component, preferably afibril component present in amyloid deposits characteristic of thedisease to be treated. In general, such antibodies are selected fortheir abilities to specifically bind the various proteins, peptides, andcomponents described with respect to the pharmaceutical compositions andmethods described in the preceding paragraphs of this section. Accordingto a related embodiment, such methods and compositions may includecombinations of antibodies that bind at least two amyloid fibrilcomponents. In general, pharmaceutical compositions are administered toprovide a serum amount of immunoreactivity against the target amyloidcomponent that is at least about four times higher than a serum level ofimmunoreactivity against the component measured in a control serumsample. The antibodies may also be administered with a carrier, asdescribed herein. In general, in accordance with this aspect of theinvention, such antibodies, will be administered (or formulated foradministration) peritoneally, orally, intranasally, subcutaneously,intramuscularly, topically or intravenously, but can be administered orformulated for administration by any pharmaceutically effective route(i.e., effective to produce the indicated therapeutic levels, as setforth above and herein).

According to a related embodiment, therapeutic antibodies may beadministered by administering a polynucleotide encoding at least oneantibody chain to the patient. According to this aspect of theinvention, the polynucleotide is expressed in the patient to produce theantibody chain in a pharmaceutically effective amount in the patient.Such a polynucleotide may encode heavy and light chains of the antibody,thereby producing the heavy and light chains in the patient.

According to preferred embodiments, the immunization regimens describedabove may include administration of agents, including antibodies, inmultiple dosages, such as over a 6 month period, such as an initialimmunization followed by booster injections at time intervals, such as 6week intervals, according to methods known in the art, or according topatient need, as assessed by immunological response. Alternatively, orin addition, such regiments may include the use of “sustained release”formulations, such as are known in the art.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Antibody titer in transgenic mice after injection with Aβ1-42.

FIG. 2: Amyloid burden in the hippocampus. The percentage of the area ofthe hippocampal region occupied by amyloid plaques, defined byreactivity with the Aβ-specific monoclonal antibody 3D6, was determinedby computer-assisted quantitative image analysis of immunoreacted brainsections. The values for individual mice are shown sorted by treatmentgroup. The horizontal line for each grouping indicates the median valueof the distribution.

FIG. 3: Neuritic dystrophy in the hippocampus. The percentage of thearea of the hippocampal region occupied by dystrophic neurites, definedby their reactivity with the human APP-specific monoclonal 8E5, wasdetermined by quantitative computer-assisted image analysis ofimmunoreacted brain sections. The values for individual mice are shownfor the AN1792-treated group and the PBS-treated control group. Thehorizontal line for each grouping indicates the median value of thedistribution.

FIG. 4: Astrocytosis in the retrosplenial cortex. The percentage of thearea of the cortical region occupied by glial fibrillary acidic protein(GFAP)-positive astrocytes was determined by quantitativecomputer-assisted image analysis of immunoreacted brain sections. Thevalues for individual mice are shown sorted by treatment group andmedian group values are indicated by horizontal lines.

FIG. 5: Geometric mean antibody titers to Aβ42 following immunizationwith a range of eight doses of Aβ42 (“AN1792”) containing 0.14, 0.4,1.2, 3.7, 11, 33, 100, or 300 μg.

FIG. 6: Kinetics of antibody response to AN1792 immunization. Titers areexpressed as geometric means of values for the 6 animals in each group.

FIG. 7: Quantitative image analysis of the cortical amyloid burden inPBS- and AN1792-treated mice.

FIG. 8: Quantitative image analysis of the neuritic plaque burden inPBS- and AN1792-treated mice.

FIG. 9: Quantitative image analysis of the percent of the retrosplenialcortex occupied by astrocytosis in PBS- and AN1792-treated mice.

FIG. 10: Lymphocyte Proliferation Assay on spleen cells fromAN1792-treated (upper panel) or PBS-treated (lower panel) mice.

FIG. 11: Total Aβ levels in the cerebral cortex. A scatterplot ofindividual Aβ profiles in mice immunized with Aβ or APP derivativescombined with Freund's adjuvant.

FIG. 12: Amyloid burden in the cortex, determined by quantitative imageanalysis of immunoreacted brain sections for mice immunized with the Aβpeptide conjugates Aβ1-5, Aβ1-12, and Aβ13-28; the full length Aβaggregates Aβ42 (“AN1792”) and Aβ1-40 (“AN1528”) and the PBS-treatedcontrol group.

FIG. 13: Geometric mean titers of Aβ-specific antibody for groups ofmice immunized with Aβ or APP derivatives combined with Freund'sadjuvant.

FIG. 14: Geometric mean titers of Aβ-specific antibody for groups ofguinea pigs immunized with AN1792, or a palmitoylated derivativethereof, combined with various adjuvants.

FIG. 15 (A-E): Aβ levels in the cortex of 12-month old PDAPP micetreated with AN1792 or AN1528 with different adjuvants.

FIG. 16: Mean titer of mice treated with polyclonal antibody to Aβ.

FIG. 17: Mean titer of mice treated with monoclonal antibody 10D5 to Aβ.

FIG. 18: Mean titer of mice treated with monoclonal antibody 2F12 to Aβ.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art of the present invention.Practitioners are particularly directed to Sambrook, et al. (1989)Molecular Cloning: A Laboratory Manual (Second Edition), Cold SpringHarbor Press, Plainview, N.Y. and Ausubel, F. M., et al. (1998) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y., fordefinitions, terms of art and standard methods known in the art ofbiochemistry and molecular biology. It is understood that this inventionis not limited to the particular methodology, protocols, and reagentsdescribed, as these may be varied to produce the same result.

The term “adjuvant” refers to a compound that, when administered inconjunction with an antigen, augments the immune response to theantigen, but when administered alone does not generate an immuneresponse to the antigen. Adjuvants can augment an immune response byseveral mechanisms including lymphocyte recruitment, stimulation of Band/or T cells, and stimulation of macrophages.

“Amyloid disease” or “amyloidosis” refers to any of a number ofdisorders which have as a symptom or as part of its pathology theaccumulation or formation of amyloid plaques.

An “amyloid plaque” is an extracellular deposit composed mainly ofproteinaceous fibrils. Generally, the fibrils are composed of a dominantprotein or peptide; however, the plaque may also include additionalcomponents that are peptide or non-peptide molecules, as describedherein.

An “amyloid component” is any molecular entity that is present in anamyloid plaque including antigenic portions of such molecules. Amyloidcomponents include but are not limited to proteins, peptides,proteoglycans, and carbohydrates. A “specific amyloid component” refersto a molecular entity that is found primarily or exclusively in theamyloid plaque of interest.

An “agent” is a chemical molecule of synthetic or biological origin. Inthe context of the present invention, an agent is generally a moleculethat can be used in a pharmaceutical composition.

An “anti-amyloid agent” is an agent which is capable of producing animmune response against an amyloid plaque component in a vertebratesubject, when administered by active or passive immunization techniques.

The terms “polynucleotide” and “nucleic acid,” as used interchangeablyherein refer to a polymeric molecule having a backbone that supportsbases capable of hydrogen bonding to typical polynucleotides, where thepolymer backbone presents the bases in a manner to permit such hydrogenbonding in a sequence specific fashion between the polymeric moleculeand a typical polynucleotide (e.g., single-stranded DNA). Such bases aretypically inosine, adenosine, guanosine, cytosine, uracil and thymidine.Polymeric molecules include double and single stranded RNA and DNA, andbackbone modifications thereof, for example, methylphosphonate linkages.

The term “polypeptide” as used herein refers to a compound made up of asingle chain of amino acid residues linked by peptide bonds. The term“protein” may be synonymous with the term “polypeptide” or may refer toa complex of two or more polypeptides.

The term “peptide” also refers to a compound composed of amino acidresidues linked by peptide bonds. Generally peptides are composed of 100or fewer amino acids, while polypeptides or proteins have more than 100amino acids. As used herein, the term “protein fragment” may also beread to mean a peptide.

A “fibril peptide” or “fibril protein” refers to a monomeric oraggregated form of a protein or peptide that forms fibrils present inamyloid plaques. Examples of such peptides and proteins are providedherein.

A “pharmaceutical composition” refers to a chemical or biologicalcomposition suitable for administration to a mammalian individual. Suchcompositions may be specifically formulated for administration via oneor more of a number of routes, including but not limited to, oral,parenteral, intravenous, intraarterial, subcutaneous, intranasal,sublingual, intraspinal, intracerebroventricular, and the like.

A “pharmaceutical excipient” or a “pharmaceutically acceptableexcipient” is a carrier, usually a liquid, in which an activetherapeutic agent is formulated. The excipient generally does notprovide any pharmacological activity to the formulation, though it mayprovide chemical and/or biological stability, release characteristics,and the like. Exemplary formulations can be found, for example, inRemington's Pharmaceutical Sciences, 19^(th) Ed., Grennaro, A., Ed.,1995.

A “glycoprotein” is protein to which at least one carbohydrate chain(oligopolysaccharide) is covalently attached.

A “proteoglycan” is a glycoprotein where at least one of thecarbohydrate chains is a glycosaminoglycan, which is a long linearpolymer of repeating disaccharides in which one member of the pairusually is a sugar acid (uronic acid) and the other is an amino sugar.

The term “immunological” or “immune” or “immunogenic” response refers tothe development of a humoral (antibody mediated) and/or a cellular(mediated by antigen-specific T cells or their secretion products)response directed against an antigen in a vertebrate individual. Such aresponse can be an active response induced by administration ofimmunogen or a passive response induced by administration of antibody orprimed T-cells. A cellular immune response is elicited by thepresentation of polypeptide epitopes in association with Class I orClass II MHC molecules to activate antigen-specific CD4⁺ T helper cellsand/or CD8⁺ cytotoxic T cells. The response may also involve activationof monocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils or other components of innateimmunity. The presence of a cell-mediated immunological response can bedetermined by standard proliferation assays (CD4⁺ T cells) or CTL(cytotoxic T lymphocyte) assays known in the art. The relativecontributions of humoral and cellular responses to the protective ortherapeutic effect of an immunogen can be distinguished by separatelyisolating immunoglobulin (IgG) and T-cell fractions from an immunizedsyngeneic animal and measuring protective or therapeutic effect in asecond subject.

An “immunogenic agent” or “immunogen” or “antigen” is a molecule that iscapable of inducing an immunological response against itself uponadministration to a patient, either in conjunction with, or in theabsence of, an adjuvant. Such molecules include, for example, amyloidfibril peptides or fragments thereof conjugated to a carrier protein,such keyhole limpet hemocyanin, Cd3 or tetanus toxin.

An “epitope” or “antigenic determinate” is the part of an antigen thatbinds to the antigen-binding region of an antibody.

The term “Aβ,” “Aβ peptide” and “Amyloid β” peptide are synonymous, andrefer to one or more peptide compositions of about 38-43 amino acidsderived from Beta Amyloid Precursor Protein (β-APP), as describedherein. “Aβxx” refers to amyloid β peptide 1-xx, where xx is a numberindicating the number of amino acids in the peptide; e.g., Aβ42 is thesame as Aβ1-42, which is also referred to herein as “AN1792,” and Aβ40is the same as Aβ1-40, which is also referred to herein as “AN1578.”

Disaggregated or monomeric Aβ means soluble, monomeric peptide units ofAβ. One method to prepare monomeric Aβ is to dissolve lyophilizedpeptide in neat DMSO with sonication. The resulting solution iscentrifuged to remove any insoluble particulates. Aggregated Aβ is amixture of oligomers in which the monomeric units are held together bynoncovalent bonds.

The term “naked polynucleotide” refers to a polynucleotide not complexedwith colloidal materials. Naked polynucleotides are sometimes cloned ina plasmid vector.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

The terms “significantly different than,” “statistically significant,”“significantly higher (or lower) than,” and similar phrases refer tocomparisons between data or other measurements, wherein the differencesbetween two compared individuals or groups are evidently or reasonablydifferent to the trained observer, or statistically significant (if thephrase includes the term “statistically” or if there is some indicationof statistical test, such as a p-value, or if the data, when analyzed,produce a statistical difference by standard statistical tests known inthe art).

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises a fibril component peptide encompasses boththe isolated peptide and the peptide as a component of a largerpolypeptide sequence. By way of further example, a composition thatcomprises elements A and B also encompasses a composition consisting ofA, B and C.

B. Amyloid Diseases

1. Overview and Pathogenesis

Amyloid diseases or amyloidoses include a number of disease stateshaving a wide variety of outward symptoms. These disorders have incommon the presence of abnormal extracellular deposits of proteinfibrils, known as “amyloid deposits” or “amyloid plaques” that areusually about 10-100 μm in diameter and are localized to specific organsor tissue regions. Such plaques are composed primarily of a naturallyoccurring soluble protein or peptide. These insoluble deposits arecomposed of generally lateral aggregates of fibrils that areapproximately 10-15 nm in diameter. Amyloid fibrils produce acharacteristic apple green birefringence in polarized light, whenstained with Congo Red dye. The disorders are classified on the basis ofthe major fibril components forming the plaque deposits, as discussedbelow.

The peptides or proteins forming the plaque deposits are often producedfrom a larger precursor protein. More specifically, the pathogenesis ofamyloid fibril deposits generally involves proteolytic cleavage of an“abnormal” precursor protein into fragments. These fragments generallyaggregate into anti-parallel β pleated sheets; however, certainundegraded forms of precursor protein have been reported to aggregateand form fibrils in familial amyloid polyneuropathy (varianttransthyretin fibrils) and dialysis-related amyloidosis (β₂microglobulin fibrils) (Tan, et al., 1994, supra).

2. Clinical Syndromes

This section provides descriptions of major types of amyloidoses,including their characteristic plaque fibril compositions. It is ageneral discovery of the present invention that amyloid diseases can betreated by administering agents that serve to stimulate an immuneresponse against a component or components of the variousdisease-specific amyloid deposits. As discussed in more detail inSection C below, such components are preferably constituents of thefibrils that form the plaques. The sections below serve to exemplifymajor forms of amyloidosis and are not intended to limit the invention.

a. AA (Reactive) Amyloidosis

Generally, AA amyloidosis is a manifestation of a number of diseasesthat provoke a sustained acute phase response. Such diseases includechronic inflammatory disorders, chronic local or systemic microbialinfections, and malignant neoplasms.

AA fibrils are generally composed of 8000 dalton fragments (AA peptideor protein) formed by proteolytic cleavage of serum amyloid A protein(apoSSA), a circulating apolipoprotein which is present in HDL particlesand which is synthesized in hepatocytes in response to such cytokines asIL-1, IL-6 and TNF. Deposition can be widespread in the body, with apreference for parenchymal organs. The spleen is usually a depositionsite, and the kidneys may also be affected. Deposition is also common inthe heart and gastrointestinal tract.

AA amyloid diseases include, but are not limited to inflammatorydiseases, such as rheumatoid arthritis, juvenile chronic arthritis,ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter'ssyndrome, Adult Still's disease, Behçet's syndrome, and Crohn's disease.AA deposits are also produced as a result of chronic microbialinfections, such as leprosy, tuberculosis, bronchiectasis, decubitusulcers, chronic pyelonephritis, osteomyelitis, and Whipple's disease.Certain malignant neoplasms can also result in AA fibril amyloiddeposits. These include such conditions as Hodgkin's lymphoma, renalcarcinoma, carcinomas of gut, lung and urogenital tract, basal cellcarcinoma, and hairy cell leukemia.

b. AL Amyloidoses

AL amyloid deposition is generally associated with almost any dyscrasiaof the B lymphocyte lineage, ranging from malignancy of plasma cells(multiple myeloma) to benign monoclonal gammopathy. At times, thepresence of amyloid deposits may be a primary indicator of theunderlying dyscrasia.

Fibrils of AL amyloid deposits are composed of monoclonal immunoglobulinlight chains or fragments thereof. More specifically, the fragments arederived from the N-terminal region of the light chain (kappa or lambda)and contain all or part of the variable (V_(L)) domain thereof. Depositsgenerally occur in the mesenchymal tissues, causing peripheral andautonomic neuropathy, carpal tunnel syndrome, macroglossia, restrictivecardiomyopathy, arthropathy of large joints, immune dyscrasias,myelomas, as well as occult dyscrasias. However, it should be noted thatalmost any tissue, particularly visceral organs such as the heart, maybe involved.

c. Hereditary Systemic Amyloidoses

There are many forms of hereditary systemic amyloidoses. Although theyare relatively rare conditions, adult onset of symptoms and theirinheritance patterns (usually autosomal dominant) lead to persistence ofsuch disorders in the general population. Generally, the syndromes areattributable to point mutations in the precursor protein leading toproduction of variant amyloidogenic peptides or proteins. Table 2summarizes the fibril composition of exemplary forms of these disorders.

TABLE 2 Hereditary Amyloidoses^(a) Fibril Peptide/Protein Geneticvariant Clinical Syndrome Transthyretin and fragments Met30, many othersFamilial amyloid (ATTR) polyneuropathy (FAP), (mainly peripheral nerves)Transthyretin and fragments Thr45, Ala60, Ser84, Cardiac involvement(ATTR) Met111, Ile122 predominant without neuropathy N-terminal fragmentof Arg 26 Familial amyloid Apolipoprotein A1 (apoAI) polyneuropathy(FAP), (mainly peripheral nerves) N-terminal fragment of Arg26, Arg50,Arg Ostertag-type, non-neuropathic Apolipoprotein A1 60, others(predominantly visceral (AapoAI) involvement) Lysozyme (Alys) Thr56,His67 Ostertag-type, non-neuropathic (predominantly visceralinvolvement) Fibrogen α chain fragment Leu554, Val 526 Ostertag-type,non-neuropathic (predominantly visceral involvement) Gelsolin fragment(Agel) Asn187, Tyr187 Cranial neuropathy with lattice corneal dystrophyCystatin C fragment Glu68 Hereditary cerebral hemorrhage (cerebralamyloid angiopathy) - Icelandic type β-amyloid protein (Aβ) Gln693Hereditary cerebral derived from Amyloid hemorrhage (cerebral amyloidPrecursor Protein (APP) angiopathy) - Dutch type β-amyloid protein (Aβ)Ile717, Phe717, Familial Alzheimer's Disease derived from Amyloid Gly717Precursor Protein (APP) β-amyloid protein (Aβ) Asn670, Leu671 FamilialDementia - probable derived from Amyloid Alzheimer's Disease PrecursorProtein (APP) Prion Protein (PrP) derived Leu102, Val167, FamilalCreutzfeldt-Jakob from PrP precursor protein Asn178, Lys200 disease;Gerstmann-Sträussler- 51-91 insert Scheinker syndrome (hereditaryspongiform encephalopathies, prion diseases) AA derived from SerumFamilal Mediterranean fever, amyloid A protein predominant renalinvolvement (ApoSSA) (autosomal recessive) AA derived from SerumMuckle-Well's syndrome, amyloid A protein (ApoSSA) nephropathy,deafness, urticaria, limb pain Unknown Cardiomyopathy with persistentatrial standstill Unknown Cutaneous deposits (bullous, papular,pustulodermal) ^(a)Data derived from Tan & Pepys, 1994, supra.

The data provided in Table 2 are exemplary and are not intended to limitthe scope of the invention. For example, more than 40 separate pointmutations in the transthyretin gene have been described, all of whichgive rise to clinically similar forms of familial amyloidpolyneuropathy.

Transthyretin (TTR) is a 14 kilodalton protein that is also sometimesreferred to as prealbumin. It is produced by the liver and choroidplexus, and it functions in transporting thyroid hormones and vitamin A.At least 50 variant forms of the protein, each characterized by a singleamino acid change, are responsible for various forms of familial amyloidpolyneuropathy. For example, substitution of proline for leucine atposition 55 results in a particularly progressive form of neuropathy;substitution of methionine for leucine at position 111 resulted in asevere cardiopathy in Danish patients. Amyloid deposits isolated fromheart tissue of patients with systemic amyloidosis have revealed thatthe deposits are composed of a heterogeneous mixture of TTR andfragments thereof, collectively referred to as ATTR, the full lengthsequences of which have been characterized. ATTR fibril components canbe extracted from such plaques and their structure and sequencedetermined according to the methods known in the art (e.g., Gustavsson,A., et al., Laboratory Invest. 73: 703-708, 1995; Kametani, F., et al.,Biochem. Biophys. Res. Commun. 125: 622-628, 1984; Pras, M., et al.,PNAS 80: 539-42, 1983).

Persons having point mutations in the molecule apolipoprotein AI (e.g.,Gly→Arg26; Trp→Arg50; Leu→Arg60) exhibit a form of amyloidosis(“Ostertag type”) characterized by deposits of the proteinapolipoprotein AI or fragments thereof (AApoAI). These patients have lowlevels of high density lipoprotein (HDL) and present with a peripheralneuropathy or renal failure.

A mutation in the alpha chain of the enzyme lysozyme (e.g., Ile→Thr56 orAsp→His57) is the basis of another form of Ostertag-type non-neuropathichereditary amyloid reported in English families. Here, fibrils of themutant lysozyme protein (Alys) are deposited, and patients generallyexhibit impaired renal function. This protein, unlike most of thefibril-forming proteins described herein, is usually present in whole(unfragmented) form (Benson, M. D., et al. CIBA Fdn. Symp. 199: 104-131,1996).

β-amyloid peptide (Aβ) is a 39-43 amino acid peptide derived byproteolysis from a large protein known as beta amyloid precursor protein(βAPP). Mutations in βAPP result in familial forms of Alzheimer'sdisease, Down's syndrome and/or senile dementia, characterized bycerebral deposition of plaques composed of Aβ fibrils and othercomponents, which are described in further detail below. Known mutationsin APP associated with Alzheimer's disease occur proximate to thecleavage sites of β or γ secretase, or within Aβ. For example, position717 is proximate to the site of γ-secretase cleavage of APP in itsprocessing to Aβ, and positions 670/671 are proximate to the site ofβ-secretase cleavage. Mutations at any of these residues may result inAlzheimer's disease, presumably by causing an increase the amount of the42/43 amino acid form of Aβ generated from APP. The structure andsequence of Aβ peptides of various lengths are well known in the art.Such peptides can be made according to methods known in the art (e.g.,Glenner and Wong, Biochem Biophys. Res. Comm. 129: 885-890, 1984;Glenner and Wong, Biochem Biophys. Res. Comm. 122: 1131-1135, 1984). Inaddition, various forms of the peptides are commercially available.

Synuclein is a synapse-associated protein that resembles an alipoproteinand is abundant in neuronal cytosol and presynaptic terminals. A peptidefragment derived from α-synuclein, termed NAC, is also a component ofamyloid plaques of Alzheimer's disease. (Clayton, et al., 1998). Thiscomponent also serves as a target for immunologically-based treatmentsof the present invention, as detailed below.

Gelsolin is a calcium binding protein that binds to and fragments actinfilaments. Mutations at position 187 (e.g., Asp→Asn; Asp→Tyr) of theprotein result in a form of hereditary systemic amyloidosis, usuallyfound in patients from Finland, as well as persons of Dutch or Japaneseorigin. In afflicted individuals, fibrils formed from gelsolin fragments(Agel), usually consist of amino acids 173-243 (68 kDa carboxyterminalfragment) and are deposited in blood vessels and basement membranes,resulting in corneal dystrophy and cranial neuropathy which progressesto peripheral neuropathy, dystrophic skin changes and deposition inother organs. (Kangas, H., et al. Human Mol. Genet. 5(9): 1237-1243,1996).

Other mutated proteins, such as mutant alpha chain of fibrinogen (AfibA)and mutant cystatin C (Acys) also form fibrils and producecharacteristic hereditary disorders. AfibA fibrils form depositscharacteristic of a nonneuropathic hereditary amyloid with renaldisease; Acys deposits are characteristic of a hereditary cerebralamyloid angiopathy reported in Iceland. (Isselbacher, et al., Harrison'sPrinciples of Internal Medicine, McGraw-Hill, San Francisco, 1995;Benson, et al., supra.). In at least some cases, patients with cerebralamyloid angiopathy (CAA) have been shown to have amyloid fibrilscontaining a non-mutant form of cystatin C in conjunction with betaprotein. (Nagai, A., et al. Molec. Chem. Neuropathol. 33: 63-78, 1998).

Certain forms of prion disease are now considered to be heritable,accounting for up to 15% of cases, which were previously thought to bepredominantly infectious in nature. (Baldwin, et al., in ResearchAdvances in Alzheimer's Disease and Related Disorders, John Wiley andSons, New York, 1995). In such prion disorders, patients develop plaquescomposed of abnormal isoforms of the normal prion protein (PrPc). Apredominant mutant isoform, PrP^(Sc), also referred to as AScr, differsfrom the normal cellular protein in its resistance to proteasedegradation, insolubility after detergent extraction, deposition insecondary lysosomes, post-translational synthesis, and high β-pleatedsheet content. Genetic linkage has been established for at least fivemutations resulting in Creutzfeldt-Jacob disease (CJD),Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familialinsomnia (FFI). (Baldwin) Methods for extracting fibril peptides fromscrapie fibrils, determining sequences and making such peptides areknown in the art. (e.g., Beekes, M., et al. J. Gen. Virol. 76: 2567-76,1995).

For example, one form of GSS has been linked to a PrP mutation at codon102, while telencephalic GSS segregates with a mutation at codon 117.Mutations at codons 198 and 217 result in a form of GSS in whichneuritic plaques characteristic of Alzheimer's disease contain PrPinstead of Aβ peptide. Certain forms of familial CJD have beenassociated with mutations at codons 200 and 210; mutations at codons 129and 178 have been found in both familial CJD and FFI. (Baldwin, supra).

d. Senile Systemic Amyloidosis

Amyloid deposition, either systemic or focal, increases with age. Forexample, fibrils of wild type transthyretin (TTR) are commonly found inthe heart tissue of elderly individuals. These may be asymptomatic,clinically silent, or may result in heart failure. Asymptomaticfibrillar focal deposits may also occur in the brain (Aβ), corporaamylacea of the prostate (Aβ₂ microglobulin), joints and seminalvesicles.

e. Cerebral Amyloidosis

Local deposition of amyloid is common in the brain, particularly inelderly individuals. The most frequent type of amyloid in the brain iscomposed primarily of Aβ peptide fibrils, resulting in dementia orsporadic (non-hereditary) Alzheimer's disease. In fact, the incidence ofsporadic Alzheimer's disease greatly exceeds forms shown to behereditary. Fibril peptides forming these plaques are very similar tothose described above, with reference to hereditary forms of Alzheimer'sdisease (AD).

f. Dialysis-Related Amyloidosis

Plaques composed of β₂ microglobulin (Aβ₂M) fibrils commonly develop inpatients receiving long term hemodialysis or peritoneal dialysis. β₂microglobulin is a 11.8 kilodalton polypeptide and is the light chain ofClass I MHC antigens, which are present on all nucleated cells. Undernormal circumstances, it is continuously shed from cell membranes and isnormally filtered by the kidney. Failure of clearance, such as in thecase of impaired renal function, leads to deposition in the kidney andother sites (primarily in collagen-rich tissues of the joints). Unlikeother fibril proteins, Aβ₂M molecules are generally present inunfragmented form in the fibrils. (Benson, supra).

g. Hormone-Derived Amyloidoses

Endocrine organs may harbor amyloid deposits, particularly in agedindividuals. Hormone-secreting tumors may also contain hormone-derivedamyloid plaques, the fibrils of which are made up of polypeptidehormones such as calcitonin (medullary carcinoma of the thyroid), isletamyloid polypeptide (amylin; occurring in most patients with Type IIdiabetes), and atrial natriuretic peptide (isolated atrial amyloidosis).Sequences and structures of these proteins are well known in the art.

h. Miscellaneous Amyloidoses

There are a variety of other forms of amyloid disease that are normallymanifest as localized deposits of amyloid. In general, these diseasesare probably the result of the localized production and/or lack ofcatabolism of specific fibril precursors or a predisposition of aparticular tissue (such as the joint) for fibril deposition. Examples ofsuch idiopathic deposition include nodular AL amyloid, cutaneousamyloid, endocrine amyloid, and tumor-related amyloid.

C. Pharmaceutical Compositions

It is the discovery of the present invention that compositions capableof eliciting or providing an immune response directed to certaincomponents of amyloid plaques are effective to treat or preventdevelopment of amyloid diseases. In particular, according to theinvention provided herein, it is possible to prevent progression of,ameliorate the symptoms of, and/or reduce amyloid plaque burden inafflicted individuals, when an immunostimulatory dose of an anti-amyloidagent, or corresponding anti-amyloid immune reagent, is administered tothe patient. This section describes exemplary anti-amyloid agents thatproduce active, as well as passive, immune responses to amyloid plaquesand provides exemplary data showing the effect treatment using suchcompositions on amyloid plaque burden.

Generally, anti-amyloid agents of the invention are composed of aspecific plaque component, preferably a fibril forming component, whichis usually a characteristic protein, peptide, or fragment thereof, asdescribed in the previous section and exemplified below. More generally,therapeutic agents for use in the present invention produce or induce animmune response against a plaque, or more specifically, a fibrilcomponent thereof. Such agents therefore include, but are not limitedto, the component itself and variants thereof, analogs and mimetics ofthe component that induce and/or cross-react with antibodies to thecomponent, as well as antibodies or T-cells that are specificallyreactive with the amyloid component. According to an important feature,pharmaceutical compositions are not selected from non-specificcomponents—that is, from those components that are generally circulatingor that are ubiquitous throughout the body. By way of example, SerumAmyloid Protein (SAP) is a circulating plasma glycoprotein that isproduced in the liver and binds to most known forms of amyloid deposits.Therapeutic compositions are preferably directed to this component.

Induction of an immune response can be active, as when an immunogen isadministered to induce antibodies or T-cells reactive with the componentin a patient, or passive, as when an antibody is administered thatitself binds to the amyloid component in the patient. Exemplary agentsfor inducing or producing an immune response against amyloid plaques aredescribed in the sections below.

Pharmaceutical compositions of the present invention may include, inaddition to the immunogenic agent(s), an effective amount of an adjuvantand/or an excipient. Pharmaceutically effective an useful adjuvants andexcipients are well known in the art, and are described in more detailin the Sections that follow.

1. Immunostimulatory Agents (Active Immune Response)

a. Anti-Fibril Compositions

One general class of preferred anti-amyloid agents consists of agentsthat are derived from amyloid fibril proteins. As mentioned above, thehallmark of amyloid diseases is the deposition in an organ or organ ofamyloid plaques consisting mainly of fibrils, which, in turn, arecomposed of characteristic fibril proteins or peptides. According to thepresent invention, such a fibril protein or peptide component is auseful agent for inducing an anti-amyloid immune response.

Tables 1 and 2 summarize exemplary fibril-forming proteins that arecharacteristic of various amyloid diseases. In accordance with thisaspect of the present invention, administration to an afflicted orsusceptible individual of an immunostimulatory composition whichincludes the appropriate fibril protein or peptide, including homologsor fragments thereof, provides therapeutic or prophylaxis with respectto the amyloid disease.

By way of example, Aβ, also known as β-amyloid peptide, or A4 peptide(see U.S. Pat. No. 4,666,829; Glenner & Wong, Biochem. Biophys. Res.Commun. 120, 1131 (1984)), is a peptide of 39-43 amino acids, which isthe principal component of characteristic plaques of Alzheimer'sdisease. Aβ is generated by processing of a larger protein APP by twoenzymes, termed β and gamma secretases (see Hardy, TINS 20, 154 (1997)).

Example I describes the results of experiments carried out in support ofthe present invention, in which Aβ42 peptide was administered toheterozygote transgenic mice that overexpress human APP with a mutationat position 717. These mice, known as “PDAPP mice” exhibitAlzheimer's-like pathology and are considered to be an animal model forAlzheimer's disease (Games, et al., Nature 373: 523-7, 1995). Asdetailed in the Example, these mice exhibit detectable Aβ plaqueneuropathology in their brains beginning at about 6 months of age, withplaque deposition progressing over time. In the experiments describedherein, aggregated Aβ42 (AN1792) was administered to the mice. Most ofthe treated mice (7/9) had no detectable amyloid in their brains at 13months of age, in contrast to control mice (saline-injected oruntreated), all of which showed significant brain amyloid burden at thisage (FIG. 2). These differences were even more pronounced in thehippocampus (FIG. 3). Treated mice also exhibited significant serumantibody titers against Aβ (all greater than 1:1000, 8/9 greater than1/10,000; FIG. 1, Table 3A). Generally, saline-treated mice exhibitedless than 4-5 times background levels of antibodies against Aβ at adilution of 1:100 at all times tested, and were therefore deemed to haveno significant response relative to control (Table 3B). These studiesdemonstrated that injection with the specific fibril forming peptide Aβprovides protection against deposition of Aβ amyloid plaques.

Serum Amyloid Protein (SAP), is a circulating plasma glycoprotein thatis produced in the liver and binds in a calcium-dependent manner to allforms of amyloid fibril, including fibrils of cerebral amyloid plaquesin Alzheimer's disease. As part of the foregoing experiments, a group ofmice was injected with SAP; these mice developed significant serumtiters to SAP (1:1000-1:30000), but did not develop detectable serumtiters to Aβ peptide and developed cerebral plaque neuropathology (FIG.2).

Further experiments, detailed in Example II, demonstrate dose dependenceof the immunogenic effect of Aβ injections in mice treated between 5weeks and about 8 months of age. In these mice, mean serum titers ofanti-Aβ peptide antibodies increased with the number of immunizationsand with increasing dosages; however, after four immunizations, serumtiters measured five days following the immunization leveled off overthe higher doses (1-300 μg) at levels around 1:10000 (FIG. 5).

Additional experiments in support of the present invention are describedin Example III, in which PDAPP model mice were treated with Aβ42commencing at a time point (about 11 months of age) after amyloidplaques were already present in their brains. In these studies, theanimals were immunized with Aβ42 or saline, and were sacrificed foramyloid burden testing at age 15 or 18 months. As illustrated in FIG. 7,at 18 months of age, Aβ42-treated mice exhibited a significantly lowermean amyloid plaque burden (plaque burden, 0.01%) than eitherPBS-treated 18-month old controls (plaque burden, 4.7%) or 12 monthuntreated animals (0.28%), where plaque burden is measured by imageanalysis, as detailed in Example XIII, part 8. These experimentsdemonstrate the efficacy of the treatment methods of the presentinvention in reducing existing plaque burden and preventing progressionof plaque burden in diseased individuals.

According to this aspect of the invention, therapeutic agents arederived from fibril peptides or proteins which comprise the plaques thatare characteristic of the disease of interest. Alternatively, suchagents are antigenically similar enough to such components to induce animmune response that also cross-reacts with the fibril component. Tables1 and 2 provide examples of such fibril peptides and proteins, thecompositions and sequences of which are known in the art or can beeasily determined according to methods known in the art. (See referencescited below and in Section B2 for references that specifically teachmethods for extraction and/or compositions of various fibril peptidecomponents; further exemplary fibril components are described below.)

Thus, in accordance with the present invention, where a diagnosis of anamyloid disease is made, based on clinical and/or biopsy determinations,the skilled practitioner will be able to ascertain the fibrilcomposition of the amyloid deposits and provide an agent that induces animmune response directed to the fibrillar peptides or proteins.

By way of example, as described above, the therapeutic agent used intreating Alzheimer's disease or other amyloid diseases characterized byAβ fibril deposition can be any of the naturally occurring forms of Aβpeptide, and particularly the human forms (i.e., Aβ39, Aβ40, Aβ41, Aβ42or Aβ43). The sequences of these peptides and their relationship to theAPP precursor are known in the art and are well known in the art (e.g.,Hardy et al., TINS 20, 155-158 (1997)). For example, Aβ42 has thesequence:

(SEQ ID NO: 1) H2N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-IIe-Ala-OH.

Aβ41, Aβ40 and Aβ39 differ from Aβ42 by the omission of Ala, Ala-Ile,and Ala-Ile-Val respectively from the C-terminal end of the peptide.Aβ43 differs from Aβ42 by the presence of a threonine residue at theC-terminus. According to a preferred embodiment of the invention,therapeutic agents will induce an immune response against all or aportion of the fibril component of the disease of interest. For example,a preferred Aβ immunogenic composition is an agent that induces anantibody specific to the free N-terminus of Aβ. Such a composition hasthe advantage that it would not recognize the precursor protein, β-APP,thereby rendering it less likely to produce autoimmunity.

By way of further example, it is appreciated that patients afflictedwith diseases characterized by the deposition of AA fibrils, forexample, certain chronic inflammatory disorders, chronic local orsystemic microbial infections, and malignant neoplasms, as describedabove, can be treated with AA peptide, a known 8 kilodalton fragment ofserum amyloid A protein (ApoSSA). Exemplary AA amyloid disordersinclude, but are not limited to inflammatory diseases such as rheumatoidarthritis, juvenile chronic arthritis, ankylosing spondylitis,psoriasis, psoriatic arthropathy, Reiter's syndrome, Adult Still'sdisease, Behçet's syndrome, Crohn's disease, chronic microbialinfections such as leprosy, tuberculosis, bronchiectasis, decubitusulcers, chronic pyelonephritis, osteomyelitis, and Whipple's disease, aswell as malignant neoplasms such as Hodgkin's lymphoma, renal carcinoma,carcinomas of gut, lung and urogenital tract, basal cell carcinoma, andhairy cell leukemia.

AA peptide refers to one or more of a heterogeneous group of peptidesderived from the N-terminus of precursor protein serum amyloid A(ApoSSA), commencing at residue 1, 2 or 3 of the precursor protein andending at any point between residues 58 and 84; commonly AA fibrils arecomposed of residues 1-76 of ApoSSA. Precise structures and compositionscan be determined, and appropriate peptides synthesized according tomethods well known in the art (Liepnieks, J. J., et al. Biochem. BiophysActa 1270: 81-86, 1995).

By way of further example, fragments derived from the N-terminal regionwhich contain all or part of the variable (V_(L)) domain ofimmunoglobulin light chains (kappa or lambda chain) generally compriseamyloid deposits in mesenchymal tissues, causing peripheral andautonomic neuropathy, carpal tunnel syndrome, macroglossia, restrictivecardiomyopathy, arthropathy of large joints, immune dyscrasias,myelomas, as well as occult dyscrasias. Compositions of the inventionwill preferably induce an immune response against a portion of the lightchain, preferably against a “neoepitope”—an epitope that is formed as aresult of fragmentation of the parent molecule—to reduce possibleautoimmune effects.

Various hereditary amyloid diseases are also amenable to the treatmentmethods of the present invention. Such diseases are described in SectionB.2, above. For example, various forms of familial amyloidpolyneuropathy are the result of at least fifty mutant forms oftransthyretin (TTR), a 14 kilodalton protein produced by the liver, eachcharacterized by a single amino acid change. While many of these formsof this disease are distinguishable on the basis of their particularpathologies and/or demographic origins, it is appreciated thattherapeutic compositions may also be composed of agents that induce animmune response against more than one form of TTR, such as a mixture oftwo or more forms of ATTR, including wildtype TTR, to provide agenerally useful therapeutic composition.

AapoAI-containing amyloid deposits are found in persons having pointmutations in the molecule apolipoprotein AI. Patients with this form ofdisease generally present with peripheral neuropathy or renal failure.According to the present invention, therapeutic compositions are made upone or more of the various forms of AapoAI described herein or known inthe art.

Certain familial forms of Alzheimer's disease, as well as Down'ssyndrome, are the result of mutations in beta amyloid precursor protein,resulting in deposition of plaques having fibrils composed mainly ofβ-amyloid peptide (Aβ). The use of Aβ peptide in therapeuticcompositions of the present invention is described above and exemplifiedherein.

Other formulations for treating hereditary forms of amyloidosis,discussed above, include compositions that produce immune responsesagainst gelsolin fragments for treatment of hereditary systemicamyloidosis, mutant lysozyme protein (Alys), for treatment of ahereditary neuropathy, mutant alpha chain of fibrinogen (AfibA) for anon-neuropathic form of amyloidosis manifest as renal disease, mutantcystatin C (Acys) for treatment of a form of hereditary cerebralangiopathy reported in Iceland. In addition, certain hereditary forms ofprion disease (e.g., Creutzfeldt-Jacob disease (CJD),Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familialinsomnia (FFI)) are characterized by a mutant isoform of prion protein,PrP^(Sc). This protein can be used in therapeutic compositions fortreatment and prevention of deposition of PrP plaques, in accordancewith the present invention.

As discussed above, amyloid deposition, either systemic or focal, isalso associated with aging. It is a further aspect of the presentinvention that such deposition can be prevented or treated byadministering to susceptible individuals compositions consisting of oneor more proteins associated with such aging. Thus, plaques composed ofATTR derived from wild type TTR are frequently found in heart tissue ofthe elderly. Similarly, certain elderly individuals may developasymptomatic fibrillar focal deposits of Aβ in their brains; Aβ peptidetreatment, as detailed herein may be warranted in such individuals. β₂microglobulin is a frequent component of corpora amylacea of theprostate, and is therefore a further candidate agent in accordance withthe present invention.

By way of further example, but not limitation, there are a number ofadditional, non-hereditary forms amyloid disease that are candidates fortreatment methods of the present invention. β₂ microglobulin fibrillarplaques commonly develop in patients receiving long term hemodialysis orperitoneal dialysis. Such patients may be treated by treatment withtherapeutic compositions directed to β2 microglobulin or, morepreferably, immunogenic epitopes thereof, in accordance with the presentinvention.

Hormone-secreting tumors may also contain hormone-derived amyloidplaques, the composition of which are generally characteristic of theparticular endocrine organ affected. Thus such fibrils may be made up ofpolypeptide hormones such as calcitonin (medullary carcinoma of thethyroid), islet amyloid polypeptide (occurring in most patients withType II diabetes), and atrial natriuretic peptide (isolated atrialamyloidosis). Compositions directed at amyloid deposits which form inthe aortic intima in atherosclerosis are also contemplated by thepresent invention. For example, Westermark, et al. describe a 69 aminoacid N-terminal fragment of Apolipoprotein A which forms such plaques(Westermark, et al. Am. J. Path. 147: 1186-92, 1995); therapeuticcompositions of the present invention include immunological reagentsdirected to such a fragment, as well as the fragment itself.

The foregoing discussion has focused on amyloid fibril components thatmay be used as therapeutic agents in treating or preventing variousforms of amyloid disease. The therapeutic agent can also be an activefragment or analog of a naturally occurring or mutant fibril peptide orprotein that contains an epitope that induces a similar protective ortherapeutic immune response on administration to a human. Immunogenicfragments typically have a sequence of at least 3, 5, 6, 10 or 20contiguous amino acids from a natural peptide. Exemplary Aβ peptideimmunogenic fragments include Aβ1-5, 1-6, 1-7, 1-10, 3-7, 1-3, 1-4,1-12, 13-28, 17-28, 1-28, 25-35, 35-40 and 35-42. Fragments lacking atleast one, and sometimes at least 5 or 10 C-terminal amino acid presentin a naturally occurring forms of the fibril component are used in somemethods. For example, a fragment lacking 5 amino acids from theC-terminal end of Aβ43 includes the first 38 amino acids from theN-terminal end of AB. Fragments from the N-terminal half of Aβ arepreferred in some methods. Analogs include allelic, species and inducedvariants. Analogs typically differ from naturally occurring peptides atone or a few positions, often by virtue of conservative substitutions.Analogs typically exhibit at least 80 or 90% sequence identity withnatural peptides. Some analogs also include unnatural amino acids ormodifications of N or C terminal amino acids. Examples of unnaturalamino acids are α,α-disubstituted amino acids, N-alkyl amino acids,lactic acid, 4-hydroxyproline, γ-carboxyglutamate,γ-N,N,N-trimethyllysine, γ-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,ω-N-methylarginine.

Generally, persons skilled in the art will appreciate that fragments andanalogs designed in accordance with this aspect of the invention can bescreened for cross-reactivity with the naturally occurring fibrilcomponents and/or prophylactic or therapeutic efficacy in transgenicanimal models as described below. Such fragments or analogs may be usedin therapeutic compositions of the present invention, if theirimmunoreactivity and animal model efficacy is roughly equivalent to orgreater than the corresponding parameters measured for the amyloidfibril components.

Such peptides, proteins, or fragments, analogs and other amyloidogenicpeptides can be synthesized by solid phase peptide synthesis orrecombinant expression, according to standard methods well known in theart, or can be obtained from natural sources. Exemplary fibrilcompositions, methods of extraction of fibrils, sequences of fibrilpeptide or protein components are provided by many of the referencescited in conjunction with the descriptions of the specific fibrilcomponents provided herein. Additionally, other compositions, methods ofextracting and determining sequences are known in the art available topersons desiring to make and use such compositions. Automatic peptidesynthesizers may be used to make such compositions and are commerciallyavailable from numerous manufacturers, such as Applied Biosystems(Perkin Elmer; Foster City, Calif.), and procedures for preparingsynthetic peptides are known in the art. Recombinant expression can bein bacteria, such as E. coli, yeast, insect cells or mammalian cells;alternatively, proteins can be produced using cell free in vitrotranslation systems known in the art. Procedures for recombinantexpression are described by Sambrook et al., Molecular Cloning: ALaboratory Manual (C.S.H.P. Press, NY 2d ed., 1989). Certain peptidesand proteins are also available commercially; for example, some forms ofAβ peptide are available from suppliers such as American PeptidesCompany, Inc., Sunnyvale, Calif., and California Peptide Research, Inc.Napa, Calif.

Therapeutic agents may also be composed of longer polypeptides thatinclude, for example, the active peptide fibril fragment or analog,together with other amino acids. For example, Aβ peptide can be presentas intact APP protein or a segment thereof, such as the C-100 fragmentthat begins at the N-terminus of Aβ and continues to the end of APP.Such polypeptides can be screened for prophylactic or therapeuticefficacy in animal models as described below. The Aβ peptide, analog,active fragment or other polypeptide can be administered in associatedform (i.e., as an amyloid peptide) or in dissociated form. Therapeuticagents may also include multimers of monomeric immunogenic agents orconjugates or carrier proteins, and/or, as mentioned above, may be addedto other fibril components, in order to provide a broader range ofanti-amyloid plaque activity.

In a further variation, an immunogenic peptide, such as a fragment ofAβ, can be presented by a virus or a bacteria as part of an immunogeniccomposition. A nucleic acid encoding the immunogenic peptide isincorporated into a genome or episome of the virus or bacteria.Optionally, the nucleic acid is incorporated in such a manner that theimmunogenic peptide is expressed as a secreted protein or as a fusionprotein with an outer surface protein of a virus or a transmembraneprotein of a bacteria so that the peptide is displayed. Viruses orbacteria used in such methods should be nonpathogenic or attenuated.Suitable viruses include adenovirus, HSV, Venezuelan equine encephalitisvirus and other alpha viruses, vesicular stomatitis virus, and otherrhabdo viruses, vaccinia and fowl pox. Suitable bacteria includeSalmonella and Shigella. Fusion of an immunogenic peptide to HBsAg ofHBV is particularly suitable. Therapeutic agents also include peptidesand other compounds that do not necessarily have a significant aminoacid sequence similarity with Aβ but nevertheless serve as mimetics ofAβ and induce a similar immune response. For example, any peptides andproteins forming β-pleated sheets can be screened for suitability.Anti-idiotypic antibodies against monoclonal antibodies to Aβ or otheramyloidogenic peptides can also be used. Such anti-Id antibodies mimicthe antigen and generate an immune response to it (see EssentialImmunology (Roit ed., Blackwell Scientific Publications, Palo Alto, 6thed.), p. 181). Agents other than Aβ peptides should induce animmunogenic response against one or more of the preferred segments of Aβlisted above (e.g., 1-10, 1-7, 1-3, and 3-7). Preferably, such agentsinduce an immunogenic response that is specifically directed to one ofthese segments without being directed to other segments of Aβ.

Random libraries of peptides or other compounds can also be screened forsuitability. Combinatorial libraries can be produced for many types ofcompounds that can be synthesized in a step-by-step fashion. Suchcompounds include polypeptides, beta-turn mimetics, polysaccharides,phospholipids, hormones, prostaglandins, steroids, aromatic compounds,heterocyclic compounds, benzodiazepines, oligomeric N-substitutedglycines and oligocarbamates. Large combinatorial libraries of thecompounds can be constructed by the encoded synthetic libraries (ESL)method described in Affymax, WO 95/12608, Affymax, WO 93/06121, ColumbiaUniversity, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO95/30642 (each of which is incorporated by reference for all purposes).Peptide libraries can also be generated by phage display methods. See,e.g., Devlin, WO 91/18980.

Combinatorial libraries and other compounds are initially screened forsuitability by determining their capacity to bind to antibodies orlymphocytes (B or T) known to be specific for Aβ or other amyloidogenicpeptides such as ATTR. For example, initial screens can be performedwith any polyclonal sera or monoclonal antibody to Aβ or any otheramyloidogenic peptide of interest. Compounds identified by such screensare then further analyzed for capacity to induce antibodies or reactivelymphocytes to Aβ or other amyloidogenic peptide. For example, multipledilutions of sera can be tested on microtiter plates that have beenprecoated with fibril peptide, and a standard ELISA can be performed totest for reactive antibodies to Aβ. Compounds can then be tested forprophylactic and therapeutic efficacy in transgenic animals predisposedto an amyloidogenic disease, as described in the Examples. Such animalsinclude, for example, mice bearing a 717 mutation of APP described byGames et al., supra, and mice bearing a 670/671 Swedish mutation of APPsuch as described by McConlogue et al., U.S. Pat. No. 5,612,486 andHsiao et al., Science 274, 99 (1996); Staufenbiel et al., Proc. Natl.Acad. Sci. USA 94, 13287-13292 (1997); Sturchler-Pierrat et al., Proc.Natl. Acad. Sci. USA 94, 13287-13292 (1997); Borchelt et al., Neuron 19,939-945 (1997)). The same screening approach can be used on otherpotential agents such as fragments of Aβ, analogs of Aβ and longerpeptides including Aβ, described above.

b. Other Plaque Components

It is appreciated that immunological responses directed at other amyloidplaque components can also be effective in preventing, retarding orreducing plaque deposition in amyloid diseases. Such components may beminor components of fibrils or associated with fibrils or fibrilformation in the plaques, with the caveat that components that areubiquitous throughout the body, or relatively non-specific to theamyloid deposit, are generally less suitable for use as therapeutictargets.

It is therefore a further discovery of the present invention that agentsthat induce an immune response to specific plaque components are usefulin treating or preventing progression of amyloid diseases. This sectionprovides background on several exemplary amyloid plaque-associatedmolecules. Induction of an immune response against any of thesemolecules, alone or in combination with immunogenic therapeuticcompositions against the fibril components described above or againstany of the other non-fibril forming components described below, providesan additional anti-amyloid treatment regimen, in accordance with thepresent invention. Also forming part of the present invention arepassive immunization regimens based on such plaque components, asdescribed herein.

By way of example, synuclein is a protein that is structurally similarto apolipoproteins but is found in neuronal cytosol, particularly in thevicinity of presynaptic terminals. There are at least three forms of theprotein, termed α, β and γ synuclein. Recently, it has been shown that αand β synuclein are involved in nucleation of amyloid deposits incertain amyloid diseases, particularly Alzheimer's disease. (Clayton, D.F., et al., TINS 21(6): 249-255, 1998). More specifically, a fragment ofthe NAC domain of a and β synuclein (residues 61-95) has been isolatedfrom amyloid plaques in Alzheimer's patients; in fact this fragmentcomprises about 10% of the plaque that remains insoluble aftersolubilization with sodium dodecyl sulfate (SDS). (George, J. M., et al.Neurosci. News 1: 12-17, 1995). Further, both the full length asynuclein and the NAC fragment thereof have been reported to acceleratethe aggregation of β-amyloid peptide into insoluble amyloid in vitro.(Clayton, supra).

Additional components associated with amyloid plaques includenon-peptide components. For example, perlecan and perlecan-derivedglycosaminoglycans are large heparin sulfate proteoglycans that arepresent in Aβ-containing amyloid plaques of Alzheimer's disease andother CNS and systemic amyloidoses, including amylin plaques associatedwith diabetes. These compounds have been shown to enhance Aβ fibrilformation. Both the core protein and glycosaminoglycan chains ofperlecan have been shown to participate in binding to Aβ. Additionalglycosaminoglycans, specifically, dermatan sulfate,chondroitin-4-sulfate, and pentosan polysulfate, are commonly found inamyloid plaques of various types and have also been shown to enhancefibril formation. Dextran sulfate also has this property. Thisenhancement is significantly reduced when the molecules are de-sulfated.Immunogenic therapeutics directed against the sulfated forms ofglycosaminoglycans, including the specific glycosaminoglycansthemselves, form an additional embodiment of the present invention,either as a primary or secondary treatment. Production of suchmolecules, as well as appropriate therapeutic compositions containingsuch molecules, is within the skill of the ordinary practitioner in theart.

2. Agents Inducing Passive Immune Response

Therapeutic agents of the invention also include immune reagents, suchas antibodies, that specifically bind to fibril peptides or othercomponents of amyloid plaques. Such antibodies can be monoclonal orpolyclonal, and have binding specificities that are consonant with thetype of amyloid disease to be targeted. Therapeutic compositions andtreatment regimens may include a antibodies directed to a single bindingdomain or epitope on a particular fibril or non-fibril component of aplaque, or may include antibodies directed to two or more epitopes onthe same component or antibodies directed to epitopes on multiplecomponents of the plaque.

For example, in experiments carried out in support of the presentinvention, 8½ to 10½ month old PDAPP mice were given intraperitoneal(i.p.) injections of polyclonal anti-Aβ42 or monoclonal anti-Aβantibodies prepared against specific epitopes of Aβ peptide, or saline,as detailed in Example XI herein. In these experiments, circulatingantibody concentrations were monitored, and booster injections weregiven as needed to maintain a circulating antibody concentration ofgreater than 1:1000 with respect to the specific antigen to which theantibody was made. Reductions in total Aβ levels were observed, comparedto control, in the cortex, hippocampus and cerebellum brain regions ofantibody-treated mice; highest reductions were exhibited in mice treatedwith polyclonal antibodies in these studies.

In further experiments carried out in support of the invention, apredictive ex vivo assay (Example XIV) was used to test clearing of anantibody against a fragment of synuclein referred to as NAC. Synucleinhas been shown to be an amyloid plaque-associated protein. An antibodyto NAC was contacted with a brain tissue sample containing amyloidplaques and microglial cells. Rabbit serum was used as a control.Subsequent monitoring showed a marked reduction in the number and sizeof plaques indicative of clearing activity of the antibody.

From these data, it is apparent that amyloid plaque load associated withAlzheimer's disease and other amyloid diseases can be greatly diminishedby administration of immune reagents directed against epitopes of Aβpeptide or against the NAC fragment of synuclein, which are effective toreduce amyloid plaque load. It is further understood that a wide varietyof antibodies can be used in such compositions. Antibodies that bindspecifically to the aggregated form of Aβ without binding to thedissociated form are suitable for use in the invention, as areantibodies that bind specifically to the dissociated form withoutbinding to the aggregated form. Other suitable antibodies bind to bothaggregated and dissociated forms. Some such antibodies bind to anaturally occurring short form of Aβ (i.e., Aβ39, 40 or 41) withoutbinding to a naturally occurring long form of Aβ (i.e., Aβ42 and Aβ43).Some antibodies bind to a long form without binding to a short form.Some antibodies bind to Aβ without binding to full-length amyloidprecursor protein. Some antibodies bind to Aβ with a binding affinitygreater than or equal to about 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹.

Polyclonal sera typically contain mixed populations of antibodiesbinding to several epitopes along the length of Aβ. Monoclonalantibodies bind to a specific epitope within Aβ that can be aconformational or nonconformational epitope. Some monoclonal antibodiesbind to an epitope within residues 1-28 of Aβ (with the first N terminalresidue of natural Aβ designated 1). Other monoclonal antibodies bind toan epitope with residues 1-10 of Aβ. There are also monoclonalantibodies that bind to an epitope with residues 1-16 of Aβ. Othermonoclonal antibodies bind to an epitope with residues 1-25 of Aβ. Somemonoclonal antibodies bind to an epitope within amino acids 1-5, 5-10,10-15, 15-20, 25-30, 10-20, 20, 30, or 10-25 of Aβ. Prophylactic andtherapeutic efficacy of antibodies can be tested using the transgenicanimal model procedures described in the Examples.

More generally, from the teachings provided herein, practitioners candesign, produce and test antibodies directed to fibril proteins orpeptides characteristic of other amyloid diseases, such as the diseasesdescribed in Section 2 herein, using compositions described herein, aswell as antibodies against other amyloid components.

a. General Characteristics of Immunoglobulins

The basic antibody structural unit is known to comprise a tetramer ofsubunits. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7 (incorporated by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat, Sequences of proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987);Chothia et al., Nature 342:878-883 (1989).

b. Production of Non-Human Antibodies

The production of non-human monoclonal antibodies, e.g., murine, guineapig, rabbit or rat, can be accomplished by, for example, immunizing theanimal with a plaque component, such as Aβ or other fibril components. Alonger polypeptide comprising Aβ or an immunogenic fragment of Aβ oranti-idiotypic antibodies to an antibody to Aβ can also be used. Seee.g., Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988)(incorporated by reference for all purposes). Such an immunogen can beobtained from a natural source, by peptide synthesis or by recombinantexpression. Optionally, the immunogen can be administered fused orotherwise complexed with a carrier protein, as described below.Optionally, the immunogen can be administered with an adjuvant. Severaltypes of adjuvant can be used as described below. Complete Freund'sadjuvant followed by incomplete adjuvant is preferred for immunizationof laboratory animals. Rabbits or guinea pigs are typically used formaking polyclonal antibodies. Mice are typically used for makingmonoclonal antibodies. Antibodies are screened for specific binding tothe immunogen. Optionally, antibodies are further screened for bindingto a specific region of the immunogen. For example, in the case of Aβpeptide as immunogen, screening can be accomplished by determiningbinding of an antibody to a collection of deletion mutants of an Aβpeptide and determining which deletion mutants bind to the antibody.Binding can be assessed, for example, by Western blot or ELISA. Thesmallest fragment to show specific binding to the antibody defines theepitope of the antibody. Alternatively, epitope specificity can bedetermined by a competition assay is which a test and reference antibodycompete for binding to the component. If the test and referenceantibodies compete, then they bind to the same epitope or epitopessufficiently proximal that binding of one antibody interferes withbinding of the other.

c. Chimeric and Humanized Antibodies

Chimeric and humanized antibodies have the same or similar bindingspecificity and affinity as a mouse or other nonhuman antibody thatprovides the starting material for construction of a chimeric orhumanized antibody. Chimeric antibodies are antibodies whose light andheavy chain genes have been constructed, typically by geneticengineering, from immunoglobulin gene segments belonging to differentspecies. For example, the variable (V) segments of the genes from amouse monoclonal antibody may be joined to human constant (C) segments,such as IgG1 and IgG4. A typical chimeric antibody is thus a hybridprotein consisting of the V or antigen-binding domain from a mouseantibody and the C or effector domain from a human antibody.

Humanized antibodies have variable region framework residuessubstantially from a human antibody (termed an acceptor antibody) andcomplementarity determining regions substantially from a mouse-antibody,(referred to as the donor immunoglobulin). See, Queen et al., Proc.Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861, U.S. Pat.No. 5,693,762, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,585,089, U.S.Pat. No. 5,530,101 and Winter, U.S. Pat. No. 5,225,539 (incorporated byreference in their entirety for all purposes). The constant region(s),if present, are also substantially or entirely from a humanimmunoglobulin. The human variable domains are usually chosen from humanantibodies whose framework sequences exhibit a high degree of sequenceidentity with the murine variable region domains from which the CDRswere derived. The heavy and light chain variable region frameworkresidues can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Carter et al., WO 92/22653. Certain aminoacids from the human variable region framework residues are selected forsubstitution based on their possible influence on CDR conformationand/or binding to antigen. Investigation of such possible influences isby modeling, examination of the characteristics of the amino acids atparticular locations, or empirical observation of the effects ofsubstitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid should usually be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region,    -   (3) otherwise interacts with a CDR region (e.g. is within about        6 A of a CDR region), or    -   (4) participates in the VL-VH interface.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of the mouse donor antibody or from the equivalentpositions of more typical human immunoglobulins. Other candidates forsubstitution are acceptor human framework amino acids that are unusualfor a human immunoglobulin at that position. The variable regionframeworks of humanized immunoglobulins usually show at least 85%sequence identity to a human variable region framework sequence orconsensus of such sequences.

d. Human Antibodies

Human antibodies against Aβ are provided by a variety of techniquesdescribed below. Some human antibodies are selected by competitivebinding experiments, or otherwise, to have the same epitope specificityas a particular mouse antibody, such as one of the mouse monoclonalsdescribed in Example XI. Human antibodies can also be screened for aparticular epitope specificity by using only a fragment of Aβ as theimmunogen, and/or by screening antibodies against a collection ofdeletion mutants of Aβ.

(1) Trioma Methodology

The basic approach and an exemplary cell fusion partner, SPAZ-4, for usein this approach have been described by Oestberg et al., Hybridoma2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman etal., U.S. Pat. No. 4,634,666 (each of which is incorporated by referencein its entirety for all purposes). The antibody-producing cell linesobtained by this method are called triomas, because they are descendedfrom three cells—two human and one mouse. Initially, a mouse myelomaline is fused with a human B-lymphocyte to obtain anon-antibody-producing xenogeneic hybrid cell, such as the SPAZ-4 cellline described by Oestberg, supra. The xenogeneic cell is then fusedwith an immunized human B-lymphocyte to obtain an antibody-producingtrioma cell line. Triomas have been found to produce antibody morestably than ordinary hybridomas made from human cells.

The immunized B-lymphocytes are obtained from the blood, spleen, lymphnodes or bone marrow of a human donor. If antibodies against a specificantigen or epitope are desired, it is preferable to use that antigen orepitope thereof for immunization. Immunization can be either in vivo orin vitro. For in vivo immunization, B cells are typically isolated froma human immunized with Aβ, a fragment thereof, larger polypeptidecontaining Aβ or fragment, or an anti-idiotypic antibody to an antibodyto Aβ. In some methods, B cells are isolated from the same patient whois ultimately to be administered antibody therapy. For in vitroimmunization, B-lymphocytes are typically exposed to antigen for aperiod of 7-14 days in a media such as RPMI-1640 (see Engleman, supra)supplemented with 10% human plasma.

The immunized B-lymphocytes are fused to a xenogeneic hybrid cell suchas SPAZ-4 by well known methods. For example, the cells are treated with40-50% polyethylene glycol of MW 1000-4000, at about 37 degrees C., forabout 5-10 min. Cells are separated from the fusion mixture andpropagated in media selective for the desired hybrids (e.g., HAT or AH).Clones secreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto Aβ or a fragment thereof. Triomas producing human antibodies havingthe desired specificity are subcloned by the limiting dilution techniqueand grown in vitro in culture medium. The trioma cell lines obtained arethen tested for the ability to bind Aβ or a fragment thereof.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into standard mammalian, bacterial or yeast celllines, according to methods well known in the art.

(2) Transgenic Non-Human Mammals

Human antibodies against Aβ can also be produced from non-humantransgenic mammals having transgenes encoding at least a segment of thehuman immunoglobulin locus. Usually, the endogenous immunoglobulin locusof such transgenic mammals is functionally inactivated. Preferably, thesegment of the human immunoglobulin locus includes unrearrangedsequences of heavy and light chain components. Both inactivation ofendogenous immunoglobulin genes and introduction of exogenousimmunoglobulin genes can be achieved by targeted homologousrecombination, or by introduction of YAC chromosomes. The transgenicmammals resulting from this process are capable of functionallyrearranging the immunoglobulin component sequences, and expressing arepertoire of antibodies of various isotypes encoded by humanimmunoglobulin genes, without expressing endogenous immunoglobulingenes. The production and properties of mammals having these propertiesare described in detail by, e.g., Lonberg et al., WO93/12227 (1993);U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No.5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat.No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S.Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature 148, 1547-1553(1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741(1991) (each of which is incorporated by reference in its entirety forall purposes). Transgenic mice are particularly suitable in this regard.Anti-Aβ antibodies are obtained by immunizing a transgenic nonhumanmammal, such as described by Lonberg or Kucherlapati, supra, with Aβ ora fragment thereof. Monoclonal antibodies are prepared by, e.g., fusingB-cells from such mammals to suitable myeloma cell lines usingconventional Kohler-Milstein technology. Human polyclonal antibodies canalso be provided in the form of serum from humans immunized with animmunogenic agent. Optionally, such polyclonal antibodies can beconcentrated by affinity purification using Aβ or other immunogenamyloid peptide as an affinity reagent.

(3) Phage Display Methods

A further approach for obtaining human anti-Aβ antibodies is to screen aDNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989). For example, asdescribed for trioma methodology, such B cells can be obtained from ahuman immunized with Aβ, fragments, longer polypeptides containing Aβ orfragments or anti-idiotypic antibodies. Optionally, such B cells areobtained from a patient who is ultimately to receive antibody treatment.Antibodies binding to an epitope of the amyloid component of interest,such as Aβ or a fragment thereof are selected. Sequences encoding suchantibodies (or a binding fragments) are then cloned and amplified. Theprotocol described by Huse is rendered more efficient in combinationwith phage-display technology. See, e.g., Dower et al., WO 91/17271 andMcCafferty et al., WO 92/01047, U.S. Pat. No. 5,877,218, U.S. Pat. No.5,871,907, U.S. Pat. No. 5,858,657, U.S. Pat. No. 5,837,242, U.S. Pat.No. 5,733,743 and U.S. Pat. No. 5,565,332 (each of which is incorporatedby reference in its entirety for all purposes). In these methods,libraries of phage are produced in which members display differentantibodies on their outer surfaces. Antibodies are usually displayed asFv or Fab fragments. Phage displaying antibodies with a desiredspecificity are selected by affinity enrichment to an Aβ peptide orfragment thereof.

In a variation of the phage-display method, human antibodies having thebinding specificity of a selected murine antibody can be produced. SeeWinter, WO 92/20791. In this method, either the heavy or light chainvariable region of the selected murine antibody is used as a startingmaterial. If, for example, a light chain variable region is selected asthe starting material, a phage library is constructed in which membersdisplay the same light chain variable region (i.e., the murine startingmaterial) and a different heavy chain variable region. The heavy chainvariable regions are obtained from a library of rearranged human heavychain variable regions. A phage showing strong specific binding for thecomponent of interest (e.g., at least 10⁸ and preferably at least 10⁹M⁻¹) is selected. The human heavy chain variable region from this phagethen serves as a starting material for constructing a further phagelibrary. In this library, each phage displays the same heavy chainvariable region (i.e., the region identified from the first displaylibrary) and a different light chain variable region. The light chainvariable regions are obtained from a library of rearranged humanvariable light chain regions. Again, phage showing strong specificbinding for amyloid peptide component are selected. These phage displaythe variable regions of completely human anti-amyloid peptideantibodies. These antibodies usually have the same or similar epitopespecificity as the murine starting material.

e. Selection of Constant Region

The heavy and light chain variable regions of chimeric, humanized, orhuman antibodies can be linked to at least a portion of a human constantregion. The choice of constant region depends, in part, whetherantibody-dependent complement and/or cellular mediated toxicity isdesired. For example, isotopes IgG1 and IgG3 have complement activityand isotypes IgG2 and IgG4 do not. Choice of isotype can also affectpassage of antibody into the brain. Light chain constant regions can belambda or kappa. Antibodies can be expressed as tetramers containing twolight and two heavy chains, as separate heavy chains, light chains, asFab, Fab′F(ab′)2, and Fv, or as single chain antibodies in which heavyand light chain variable domains are linked through a spacer.

f. Expression of Recombinant Antibodies

Chimeric, humanized and human antibodies are typically produced byrecombinant expression. Recombinant polynucleotide constructs typicallyinclude an expression control sequence operably linked to the codingsequences of antibody chains, including naturally-associated orheterologous promoter regions. Preferably, the expression controlsequences are eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g.,ampicillin-resistance or hygromycin-resistance, to permit detection ofthose cells transformed with the desired DNA sequences.

E. coli is one prokaryotic host particularly useful for cloning the DNAsequences of the present invention. Microbes, such as yeast are alsouseful for expression. Saccharomyces is a preferred yeast host, withsuitable vectors having expression control sequences, an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, NY, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines, various COS celllines, HeLa cells, L cells and myeloma cell lines. Expression vectorsfor these cells can include expression control sequences, such as anorigin of replication, a promoter, an enhancer (Queen et al., Immunol.Rev. 89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from endogenous genes, cytomegalovirus,SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J.Immunol. 148:1149 (1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (e.g.,according to methods described in U.S. Pat. No. 5,741,957, U.S. Pat. No.5,304,489, U.S. Pat. No. 5,849,992, all incorporated by reference hereinin their entireties). Suitable transgenes include coding sequences forlight and/or heavy chains in operable linkage with a promoter andenhancer from a mammary gland specific gene, such as casein or betalactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment,electroporation, lipofection, biolistics or viral-based transfection canbe used for other cellular hosts. Other methods used to transformmammalian cells include the use of polybrene, protoplast fusion,liposomes, electroporation, and microinjection (see generally, Sambrooket al., supra). For production of transgenic animals, transgenes can bemicroinjected into fertilized oocytes, or can be incorporated into thegenome of embryonic stem cells, and the nuclei of such cells transferredinto enucleated oocytes.

Once expressed, antibodies can be purified according to standardprocedures of the art, including HPLC purification, columnchromatography, gel electrophoresis and the like (see generally, Scopes,Protein Purification (Springer-Verlag, NY, 1982)).

4. Other Therapeutic Agents

Therapeutic agents for use in the present methods also include T-cellsthat bind to a plaque component, such as Aβ peptide. For example,T-cells can be activated against Aβ peptide by expressing a human MHCclass I gene and a human β-2-microglobulin gene from an insect cellline, whereby an empty complex is formed on the surface of the cells andcan bind to Aβ peptide. T-cells contacted with the cell line becomespecifically activated against the peptide. See Peterson et al., U.S.Pat. No. 5,314,813. Insect cell lines expressing an MHC class II antigencan similarly be used to activate CD4 T cells.

5. Carrier Proteins

Some agents for inducing an immune response contain the appropriateepitope for inducing an immune response against amyloid deposits but aretoo small to be immunogenic. In this situation, a peptide immunogen canbe linked to a suitable carrier to help elicit an immune response.Suitable carriers include serum albumins, keyhole limpet hemocyanin,immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or atoxoid from other pathogenic bacteria, such as diphtheria, E. coli,cholera, or H. pylori, or an attenuated toxin derivative. Other carriersinclude T-cell epitopes that bind to multiple MHC alleles, e.g., atleast 75% of all human MHC alleles. Such carriers are sometimes known inthe art as “universal T-cell epitopes.” Examples of universal T-cellepitopes include:

Influenza Hemagluttinin: HA₃₀₇₋₃₁₉ PKYVKQNTLKLAT (SEQ ID NO: 1) PADRE(common residues bolded) AKXVAAWTLKAAA (SEQ ID NO: 2) Malaria CS: T3epitope EKKIAKMEKASSVFNV (SEQ ID NO: 3) Hepatitis B surface antigen:HBsAg₁₉₋₂₈ FFLLTRILTI (SEQ ID NO: 4) Heat Shock Protein 65: hsp65₁₅₃₋₁₇₁DQSIGDLIAEAMDKVGNEG (SEQ ID NO: 5) bacille Calmette-GuerinQVHFQPLPPAVVKL (SEQ ID NO: 6) Tetanus toxoid: TT₈₃₀₋₈₄₄ QYIKANSKFIGITEL(SEQ ID NO: 7) Tetanus toxoid: TT₉₄₇₋₉₆₇ FNNFTVSFWLRVPKVSASHLE (SEQ IDNO: 8) HIVgp 120 T1: KQIINMWQEVGKAMYA. (SEQ ID NO: 9)

Other carriers for stimulating or enhancing an immune response includecytokines such as IL-1, IL-1 α and β peptides, IL-2, γINF, IL-10,GM-CSF, and chemokines, such as MIP1α and β and RANTES. Immunogenicagents can also be linked to peptides that enhance transport acrosstissues, as described in O'Mahony, WO 97/17613 and WO 97/17614.

Immunogenic agents can be linked to carriers by chemical crosslinking.Techniques for linking an immunogen to a carrier include the formationof disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) (if the peptide lacks a sulfhydryl group, this can be provided byaddition of a cysteine residue). These reagents create a disulfidelinkage between themselves and peptide cysteine resides on one proteinand an amide linkage through the ε-amino on a lysine, or other freeamino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185(1982). Other bifunctional coupling agents form a thioether rather thana disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenic peptides can also be expressed as fusion proteins withcarriers (i.e., heterologous peptides). The immunogenic peptide can belinked at its amino terminus, its carboxyl terminus, or both to acarrier. Optionally, multiple repeats of the immunogenic peptide can bepresent in the fusion protein. Optionally, an immunogenic peptide can belinked to multiple copies of a heterologous peptide, for example, atboth the N and C termini of the peptide. Some carrier peptides serve toinduce a helper T-cell response against the carrier peptide. The inducedhelper T-cells in turn induce a B-cell response against the immunogenicpeptide linked to the carrier peptide.

Some agents of the invention comprise a fusion protein in which anN-terminal fragment of Aβ is linked at its C-terminus to a carrierpeptide. In such agents, the N-terminal residue of the fragment of Aβconstitutes the N-terminal residue of the fusion protein. Accordingly,such fusion proteins are effective in inducing antibodies that bind toan epitope that requires the N-terminal residue of Aβ to be in freeform. Some agents of the invention comprises a plurality of repeats ofan N-terminal segment of Aβ linked at the C-terminus to one or more copyof a carrier peptide. The N-terminal fragment of Aβ incorporated intosuch fusion proteins sometimes begins at Aβ1-3 and ends at Aβ7-11.Aβ1-7, Aβ1-3, 1-4, 1-5, and 3-7 are preferred N-terminal fragment of Aβ.Some fusion proteins comprise different N-terminal segments of Aβ intandem. For example, a fusion protein can comprise Aβ1-7 followed byAβ1-3 followed by a heterologous peptide.

In some fusion proteins, an N-terminal segment of Aβ is fused at itsN-terminal end to a heterologous carrier peptide. The same variety ofN-terminal segments of Aβ can be used as with C-terminal fusions. Somefusion proteins comprise a heterologous peptide linked to the N-terminusof an N-terminal segment of Aβ, which is in turn linked to one or moreadditional N-terminal segments of Aβ in tandem.

Some examples of fusion proteins suitable for use in the invention areshown below. Some of these fusion proteins comprise segments of Aβlinked to tetanus toxoid epitopes such as described in U.S. Pat. No.5,196,512, EP 378,881 and EP 427,347. Some fusion proteins comprisessegments of Aβ linked to carrier peptides described in U.S. Pat. No.5,736,142. Some heterologous peptides are universal T-cell epitopes. Insome methods, the agent for administration is simply a single fusionprotein with an Aβ segment linked to a heterologous segment in linearconfiguration. In some methods, the agent is multimer of fusion proteinsrepresented by the formula 2^(x), in which x is an integer from 1-5.Preferably x is 1, 2 or 3, with 2 being most preferred. When x is two,such a multimer has four fusion proteins linked in a preferredconfiguration referred to as MAP4 (see U.S. Pat. No. 5,229,490).Epitopes of Aβ are underlined.

The MAP4 configuration is shown below, where branched structures areproduced by initiating peptide synthesis at both the N terminal and sidechain amines of lysine. Depending upon the number of times lysine isincorporated into the sequence and allowed to branch, the resultingstructure will present multiple N termini. In this example, fouridentical N termini have been produced on the branched lysine-containingcore. Such multiplicity greatly enhances the responsiveness of cognate Bcells.

(SEQ ID NO: 10) AN90549 (Aβ 1-7/Tetanus toxoid 830-844 in a MAP4configuration): DAEFRHDQYIKANSKFIGITEL (SEQ ID NO: 11) AN90550 (Aβ1-7/Tetanus toxoid 947-967 in a MAP4 configuration):DAEFRHDFNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 12) AN90542 (Aβ 1-7/Tetanustoxoid 830-844 + 947-967 in a linear configuration):DAEFRHDQYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 13) AN90576: (Aβ3-9)/Tetanus toxoid 830-844 in a MAP4 configuration):EFRHDSGQYIKANSKFIGITEL (SEQ ID NO: 14) Peptide described in U.S. Pat.No. 5,736,142 (all in linear configurations): AN90562 (Aβ 1-7/peptide)AKXVAAWTLKAAADAEFRHD (SEQ ID NO: 15) AN90543 (Aβ1-7 × 3/peptide):DAEFRHDDAEFRHDDAEFRHDAKXVAAWTLKAAA (SEQ ID NO: 16) Other examples offusion proteins (immunogenic epitope of Aβ bolded) includeAKXVAAWTLKAAA-DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO: 17)DAEFRHD-AKXVAAWTLKAAA (SEQ ID NO: 18) DAEFRHD-ISQAVHAAHAEINEAGR (SEQ IDNO: 19) FRHDSGY-ISQAVHAAHAEINEAGR (SEQ ID NO: 20)EFRHDSG-ISQAVHAAHAEINEAGR (SEQ ID NO: 21)PKYVKQNTLKLAT-DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO: 22)DAEFRHD-PKYVKQNTLKLAT-DAEFRHD (SEQ ID NO: 23)DAEFRHD-DAEFRHD-DAEFRHD-PKYVKQNTLKLAT (SEQ ID NO: 24)DAEFRHD-DAEFRHD-PKYVKQNTLKLAT (SEQ ID NO: 25)DAEFRHD-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE-DAEFRHDDAEFRHD-DAEFRHD-DAEFRHD-QYIKANSKFIGITEL- FNNFTVSFWLRVPKVSASHLE (SEQ IDNO: 26) DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 27)DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE- DAEFRHD (SEQ ID NO: 28)DAEFRHD-QYIKANSKFIGITEL on a 2 branched resin

SEQ ID NO: 29) EQVTNVGGAISQAVHAAHAEINEAGR (Synuclein fusion protein inMAP-4 configuration;

The same or similar carrier proteins and methods of linkage can be usedfor generating immunogens to be used in generation of antibodies againstAβ for use in passive immunization. For example, Aβ or a fragment linkedto a carrier can be administered to a laboratory animal in theproduction of monoclonal antibodies to Aβ.

6. Nucleic Acid Encoding Therapeutic Agents

Immune responses against amyloid deposits can also be induced byadministration of nucleic acids encoding selected peptide immunogens, orantibodies and their component chains used for passive immunization.Such nucleic acids can be DNA or RNA. A nucleic acid segment encoding animmunogen is typically linked to regulatory elements, such as a promoterand enhancer, that allow expression of the DNA segment in the intendedtarget cells of a patient. For expression in blood cells, as isdesirable for induction of an immune response, promoter and enhancerelements from light or heavy chain immunoglobulin genes or the CMV majorintermediate early promoter and enhancer are suitable to directexpression. The linked regulatory elements and coding sequences areoften cloned into a vector. For administration of double-chainantibodies, the two chains can be cloned in the same or separatevectors.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3,102-109, 1993); adenoviral vectors (see, e.g., Bett et al., J. Virol.67, 5911, 1993); adeno-associated virus vectors (see, e.g., Zhou et al.,J. Exp. Med. 179, 1867, 1994), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519,1996), Venezuelan equine encephalitis virus (see U.S. Pat. No.5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (see WO96/34625) and papillomaviruses (Ohe et al., Human Gene Therapy 6,325-333, 1995); Woo et al., WO 94/12629 and Xiao & Brandsma, NucleicAcids. Res. 24, 2630-2622, 1996).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed toor associated with particulate carriers, examples of which includepolymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, intranasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application (see e.g., U.S. Pat. No. 5,399,346). Such vectorscan further include facilitating agents such as bupivacaine (U.S. Pat.No. 5,593,970). DNA can also be administered using a gene gun. See Xiao& Brandsma, supra. The DNA encoding an immunogen is precipitated ontothe surface of microscopic metal beads. The microprojectiles areaccelerated with a shock wave or expanding helium gas, and penetratetissues to a depth of several cell layers. For example, The Accel™ GeneDelivery Device manufactured by Agracetus, Inc., (Middleton, Wis.) issuitable. Alternatively, naked DNA can pass through skin into the bloodstream simply by spotting the DNA onto skin with chemical or mechanicalirritation (see WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

7. Screening Antibodies for Clearing Activity

Example XIV describes methods of screening an antibody for activity inclearing an amyloid deposit. To screen for activity against an amyloiddeposit, a tissue sample from a patient with amyloidosis, such as braintissue in Alzheimer's disease, or an animal model having characteristicamyloid pathology is contacted with phagocytic cells bearing an Fcreceptor, such as microglial cells, and the antibody under test in amedium in vitro. The phagocytic cells can be a primary culture or a cellline, such as BV-2, C8-B4, or THP-1. These components are combined on amicroscope slide to facilitate microscopic monitoring, or multiplereactions may be performed in parallel in the wells of a microtiterdish. In such a format, a separate miniature microscope slide can bemounted in the separate wells, or a nonmicroscopic detection format,such as ELISA detection of Aβ can be used. Preferably, a series ofmeasurements is made of the amount of amyloid deposit in the in vitroreaction mixture, starting from a baseline value before the reaction hasproceeded, and one or more test values during the reaction. The antigencan be detected by staining, for example, with a fluorescently labelledantibody to Aβ or other component of amyloid plaques. The antibody usedfor staining may or may not be the same as the antibody being tested forclearing activity. A reduction relative to baseline during the reactionof the amyloid deposits indicates that the antibody under test hasclearing activity. Such antibodies are likely to be useful in preventingor treating Alzheimer's and other amyloidogenic diseases. As describedabove, experiments carried out in support of the present inventionrevealed, using such an assay, that antibodies to the NAC fragment ofsynuclein are effective to clear amyloid plaques characteristic ofAlzheimer's disease.

D. Patients Amenable to Anti-Amyloid Treatment Regimens

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showing symptomsof amyloidosis. In the case of Alzheimer's disease, virtually anyone isat risk of suffering from Alzheimer's disease if he or she lives longenough. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods areespecially useful for individuals who do have a known genetic risk ofAlzheimer's disease or any of the other hereditary amyloid diseases.Such individuals include those having relatives who have experiencedthis disease, and those whose risk is determined by analysis of geneticor biochemical markers. Genetic markers of risk toward Alzheimer'sdisease include mutations in the APP gene, particularly mutations atposition 717 and positions 670 and 671 referred to as the Hardy andSwedish mutations respectively (see Hardy, TINS, supra). Other markersof risk are mutations in the presenilin genes, PS1 and PS2, and ApoE4,family history of AD, hypercholesterolemia or atherosclerosis.Individuals presently suffering from Alzheimer's disease can berecognized from characteristic dementia, as well as the presence of riskfactors described above. In addition, a number of diagnostic tests areavailable for identifying individuals who have AD. These includemeasurement of CSF tau and Aβ42 levels. Elevated tau and decreased Aβ42levels signify the presence of AD. Individuals suffering fromAlzheimer's disease can also be diagnosed by MMSE or ADRDA criteria asdiscussed in the Examples section.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to the therapeuticagent (e.g., Aβ peptide) over time, along the lines described inExamples I and II herein. If the response falls, a booster dosage isindicated. In the case of potential Down's syndrome patients, treatmentcan begin antenatally by administering therapeutic agent to the motheror shortly after birth.

Other forms of amyloidosis often go undiagnosed, unless a particularpredilection for the disease is suspected. One prime symptom is thepresence of cardiac or renal disease in a middle-aged to elderly patientwho also has signs of other organ involvement. Low voltage or extremeaxis deviations of the electrocardiogram and thickened ventriculartissue may be indicative of cardiac involvement. Proteinuria is asymptom of renal involvement. Hepatic involvement may also be suspected,if hepatomegaly is detected by physical examination of the patient.Peripheral neuropathy is also a common occurrence in certain forms ofamyloidoses; autonomic neuropathy, characterized by posturalhypotension, may also be found. Amyloidosis should be suspected inanyone with a progressive neuropathy of indeterminate origin. Adefinitive diagnosis of the disease can be made using tissue biopsymethods, where the affected organ(s) are available. For systemicamyloidoses, a fat pad aspirated or rectal biopsy samples may be used.The biopsy material is stained with Congo red, with positive samplesexhibiting apple green birefringence under polarized light microscopy.

E. Treatment Regimens

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of, aparticular disease in an amount sufficient to eliminate or reduce therisk or delay the outset of the disease. In therapeutic applications,compositions or medicants are administered to a patient suspected of, oralready suffering from such a disease in an amount sufficient to cure,or at least partially arrest, the symptoms of the disease and itscomplications. An amount adequate to accomplish this is defined as atherapeutically- or pharmaceutically-effective dose. In bothprophylactic and therapeutic regimes, agents are usually administered inseveral dosages until a sufficient immune response has been achieved.Typically, the immune response is monitored and repeated dosages aregiven if the immune response starts to wane.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human, but insome diseases, such as prion protein-associated mad cow disease, thepatient can be a nonhuman mammal, such as a bovine. Treatment dosagesneed to be titrated to optimize safety and efficacy. The amount ofimmunogen depends on whether adjuvant is also administered, with higherdosages generally being required in the absence of adjuvant. Dependingon the immunogenicity of the particular formulation, an amount of animmunogen for administration may vary from 1 μg-500 μg per patient andmore usually from 5-500 μg per injection for human administration.Occasionally, a higher dose of 0.5-5 mg per injection is used. Typicallyat least about 10, 20, 50 or 100 μg is used for each human injection.The timing of injections can vary significantly from once a day, to oncea year, to once a decade, with successive “boosts” of immunogen somewhatpreferred. Generally, in accordance with the teachings provided herein,effective dosages can be monitored by obtaining a fluid sample from thepatient, generally a blood serum sample, and determining the titer ofantibody developed against the immunogen, using methods well known inthe art and readily adaptable to the specific antigen to be measured.Ideally, a sample is taken prior to initial dosing; subsequent samplesare taken and titered after each immunization. Generally, a dose ordosing schedule which provides a detectable titer at least four timesgreater than control or “background” levels at a serum dilution of 1:100is desirable, where background is defined relative to a control serum orrelative to a plate background in ELISA assays. Titers of at least1:1000 or 1:5000 are preferred in accordance with the present invention.

On any given day that a dosage of immunogen is given, the dosage isusually greater than about 1 μg/patient and preferably greater than 10μg/patient if adjuvant is also administered, and at least greater than10 μg/patient and usually greater than 100 μg/patient in the absence ofadjuvant. Doses for individual immunogens, selected in accordance withthe present invention, are determined according to standard dosing andtitering methods, taken in conjunction with the teachings providedherein. A typical regimen consists of an immunization followed bybooster injections at time intervals, such as 6 week intervals. Anotherregimen consists of an immunization followed by booster injections 1, 2and 12 months later. Another regimen entails an injection every twomonths for life. Alternatively, booster injections can be on anirregular basis as indicated by monitoring of immune response.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight. An exemplary treatment regime entails administration once perevery two weeks or once a month or once every 3 to 6 months. In somemethods, two or more monoclonal antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody toAβ in the patient. Alternatively, antibody can be administered as asustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the antibody in the patient. In general, human antibodiesshow the longest half life, followed by humanized antibodies, chimericantibodies, and nonhuman antibodies. The dosage and frequency ofadministration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Agents for inducing an immune response can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraperitoneal,intranasal or intramuscular means for prophylactic and/or therapeutictreatment. Typical routes of administration of an immunogenic agent areintramuscular (i.m.), intravenous (i.v.) or subcutaneous (s.c.),although other routes can be equally effective. Intramuscular injectionis most typically performed in the arm or leg muscles. In some methods,agents are injected directly into a particular tissue where depositshave accumulated, for example intracranial injection. Intramuscularinjection or intravenous infusion are preferred for administration ofantibody. In some methods, particular therapeutic antibodies areinjected directly into the cranium. In some methods, antibodies areadministered as a sustained release composition or device, such as aMedipad™ device.

Agents of the invention can optionally be administered in combinationwith other agents that are at least partly effective in treatment ofamyloidogenic disease. In the case of Alzheimer's and Down's syndrome,in which amyloid deposits occur in the brain, agents of the inventioncan also be administered in conjunction with other agents that increasepassage of the agents of the invention across the blood-brain barrier.Further, therapeutic cocktails comprising immunogens designed to provokean immune response against more than one amyloid component are alsocontemplated by the present invention, as are a combination of anantibody directed against one plaque component and an immunogen directedto a different plaque component.

Immunogenic agents of the invention, such as peptides, are sometimesadministered in combination with an adjuvant. A variety of adjuvants canbe used in combination with a peptide, such as Aβ, to elicit an immuneresponse. Preferred adjuvants augment the intrinsic response to animmunogen without causing conformational changes in the immunogen thataffect the qualitative form of the response. Preferred adjuvants includealuminum hydroxide and aluminum phosphate, 3 De-O-acylatedmonophosphoryl lipid A (MPL™) (see GB 2220211 (RIBI ImmunoChem ResearchInc., Hamilton, Mont., now part of Corixa). Stimulon™ QS-21 is atriterpene glycoside or saponin isolated from the bark of the QuillajaSaponaria Molina tree found in South America (see Kensil et al., inVaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman,Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540), (AquilaBioPharmaceuticals, Framingham, Mass.). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., N. Engl. J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (WO98/40100). Alternatively, Aβ can be coupled to an adjuvant. However,such coupling should not substantially change the conformation of Aβ soas to affect the nature of the immune response thereto. Adjuvants can beadministered as a component of a therapeutic composition with an activeagent or can be administered separately, before, concurrently with, orafter administration of the therapeutic agent.

A preferred class of adjuvants is aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvantscan be used with or without other specific immunostimulating agents suchas MPL or 3-DMP, QS-21, polymeric or monomeric amino acids such aspolyglutamic acid or polylysine. Another class of adjuvants isoil-in-water emulsion formulations. Such adjuvants can be used with orwithout other specific immunostimulating agents such as muramyl peptides(e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™, or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphoryl lipid A, trehalose dimycolate (TDM), andcell wall skeleton (CWS), preferably MPL+CWS (Detox™). Another class ofpreferred adjuvants is saponin adjuvants, such as Stimulon™ (QS-21;Aquila, Framingham, Mass.) or particles generated therefrom such asISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvantsinclude Incomplete Freund's Adjuvant (IFA), cytokines, such asinterleukins (IL-1, IL-2, and IL-12), macrophage colony stimulatingfactor (M-CSF), and tumor necrosis factor (TNF). Such adjuvants aregenerally available from commercial sources.

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel indicating the intended therapeutic application. If immunogen andadjuvant are packaged separately, the packaging typically includesinstructions for mixing before use. The choice of an adjuvant and/orcarrier depends on such factors as the stability of the formulationcontaining the adjuvant, the route of administration, the dosingschedule, and the efficacy of the adjuvant for the species beingvaccinated. In humans, a preferred pharmaceutically acceptable adjuvantis one that has been approved for human administration by pertinentregulatory bodies. Examples of such preferred adjuvants for humansinclude alum, MPL and QS-21. Optionally, two or more different adjuvantscan be used simultaneously. Preferred combinations include alum withMPL, alum with QS-21, MPL with QS-21, and alum, QS-21 and MPL together.Also, Incomplete Freund's adjuvant can be used (Chang et al., AdvancedDrug Delivery Reviews 32, 173-186 (1998)), optionally in combinationwith any of alum, QS-21, and MPL and all combinations thereof.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent and a variety ofother pharmaceutically acceptable components. See Remington'sPharmaceutical Science (19th ed., 1995). The preferred form depends onthe intended mode of administration and therapeutic application. Thecompositions can also include, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249,1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).The agents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin patchor using transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24(1995); Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).

F. Methods of Diagnosis

The invention provides methods of detecting an immune response againstAβ peptide in a patient suffering from or susceptible to Alzheimer'sdisease. The methods are particularly useful for monitoring a course oftreatment being administered to a patient. The methods can be used tomonitor both therapeutic treatment on symptomatic patients andprophylactic treatment on asymptomatic patients. The methods are usefulfor monitoring both active immunization (e.g., antibody produced inresponse to administration of immunogen) and passive immunization (e.g.,measuring level of administered antibody).

1. Active Immunization

Some methods entail determining a baseline value of an immune responsein a patient before administering a dosage of agent, and comparing thiswith a value for the immune response after treatment. A significantincrease (i.e., greater than the typical margin of experimental error inrepeat measurements of the same sample, expressed as one standarddeviation from the mean of such measurements) in value of the immuneresponse signals a positive treatment outcome (i.e., that administrationof the agent has achieved or augmented an immune response). If the valuefor immune response does not change significantly, or decreases, anegative treatment outcome is indicated. In general, patients undergoingan initial course of treatment with an immunogenic agent are expected toshow an increase in immune response with successive dosages, whicheventually reaches a plateau. Administration of agent is generallycontinued while the immune response is increasing. Attainment of theplateau is an indicator that the administered of treatment can bediscontinued or reduced in dosage or frequency.

In other methods, a control value (i.e., a mean and standard deviation)of immune response is determined for a control population. Typically theindividuals in the control population have not received prior treatment.Measured values of immune response in a patient after administering atherapeutic agent are then compared with the control value. Asignificant increase relative to the control value (e.g., greater thanone standard deviation from the mean) signals a positive treatmentoutcome. A lack of significant increase or a decrease signals a negativetreatment outcome. Administration of agent is generally continued whilethe immune response is increasing relative to the control value. Asbefore, attainment of a plateau relative to control values in anindicator that the administration of treatment can be discontinued orreduced in dosage or frequency.

In other methods, a control value of immune response (e.g., a mean andstandard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose immune responses have plateaued in response to treatment. Measuredvalues of immune response in a patient are compared with the controlvalue. If the measured level in a patient is not significantly different(e.g., more than one standard deviation) from the control value,treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the level in the patient persists below the controlvalue, then a change in treatment regime, for example, use of adifferent adjuvant may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for immuneresponse to determine whether a resumption of treatment is required. Themeasured value of immune response in the patient can be compared with avalue of immune response previously achieved in the patient after aprevious course of treatment. A significant decrease relative to theprevious measurement (i.e., greater than a typical margin of error inrepeat measurements of the same sample) is an indication that treatmentcan be resumed. Alternatively, the value measured in a patient can becompared with a control value (mean plus standard deviation) determinedin a population of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

The tissue sample for analysis is typically blood, plasma, serum, mucousor cerebrospinal fluid from the patient. The sample is analyzed forindication of an immune response to the amyloid component of interest,such as any form of Aβ peptide. The immune response can be determinedfrom the presence of, e.g., antibodies or T-cells that specifically bindto the component of interest, such as Aβ peptide. ELISA methods ofdetecting antibodies specific to Aβ are described in the Examplessection and can be applied to other peptide antigens. Methods ofdetecting reactive T-cells are well known in the art.

2. Passive Immunization

In general, the procedures for monitoring passive immunization aresimilar to those for monitoring active immunization described above.However, the antibody profile following passive immunization typicallyshows an immediate peak in antibody concentration followed by anexponential decay. Without a further dosage, the decay approachespretreatment levels within a period of days to months depending on thehalf-life of the antibody administered. For example the half-life ofsome human antibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to Aβ in the patientis made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dosage of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other patients. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one standard deviation of the referencevalue in population of patients benefiting from treatment)administration of an additional dosage of antibody is indicated.

3. Diagnostic Kits

The invention further provides diagnostic kits for performing thediagnostic methods described above. Typically, such kits contain anagent that specifically binds to antibodies to an amyloid plaquecomponent, such as Aβ, or reacts with T-cells specific for thecomponent. The kit can also include a label. For detection of antibodiesto Aβ, the label is typically in the form of labelled anti-idiotypicantibodies. For detection of antibodies, the agent can be suppliedprebound to a solid phase, such as to the wells of a microtiter dish.For detection of reactive T-cells, the label can be supplied as3H-thymidine to measure a proliferative response. Kits also typicallycontain labelling providing directions for use of the kit. The labellingmay also include a chart or other correspondence regime correlatinglevels of measured label with levels of antibodies to Aβ or T-cellsreactive with Aβ. The term labelling refers to any written or recordedmaterial that is attached to, or otherwise accompanies a kit at any timeduring its manufacture, transport, sale or use. For example, the termlabelling encompasses advertising leaflets and brochures, packagingmaterials, instructions, audio or video cassettes, computer discs, aswell as writing imprinted directly on kits.

EXAMPLES I. Prophylactic Efficacy of Aβ Against Alzheimer's Disease (AD)

These examples describe administration of Aβ42 peptide to transgenicmice overexpressing APP with a mutation at position 717 (APP_(717V→F))that predisposes them to develop Alzheimer's-like neuropathology.Production and characteristics of these mice (PDAPP mice) is describedin Games et al., Nature, supra. These animals, in their heterozygoteform, begin to deposit Aβ at six months of age forward. By fifteenmonths of age they exhibit levels of Aβ deposition equivalent to thatseen in Alzheimer's disease. PDAPP mice were injected with aggregatedAβ₄₂ (aggregated Aβ₄₂) or phosphate buffered saline. Aggregated Aβ₄₂ waschosen because of its ability to induce antibodies to multiple epitopesof Aβ.

A. Methods

1. Source of Mice

Thirty PDAPP heterogenic female mice were randomly divided into thefollowing groups: 10 mice for injection with aggregated Aβ42 (one diedin transit), 5 mice to be injected with PBS/adjuvant or PBS, and 10uninjected controls. Five mice were injected with peptides derived fromthe sequence of serum amyloid protein (SAP).

2. Preparation of Immunogens

Preparation of aggregated Aβ42: two milligrams of Aβ42 (US Peptides Inc,lot K-42-12) was dissolved in 0.9 ml water and made up to 1 ml by adding0.1 ml 10×PBS. This was vortexed and allowed to incubate overnight 37°C., under which conditions the peptide aggregated. Any unused Aβ wasstored as a dry lyophilized powder at −20° C. until the next injection.

It should be noted that when such commercially available peptides areused, the dry weights may include salt weights; weights reported in allExamples herein, unless otherwise indicated, include salt weights. Exactmasses of peptide may be determined using standard assays of thepreparation, such as nitrogen determination, in conjunction with theknown composition.

3. Preparation of Injections

For each injection, 100 μg of aggregated Aβ42 in PBS per mouse wasemulsified 1:1 with Complete Freund's adjuvant (CFA) in a final volumeof 400 μl emulsion for the first immunization, followed by a boost ofthe same amount of immunogen in Incomplete Freund's adjuvant (IFA) at 2weeks. Two additional doses in IFA were given at monthly intervals. Thesubsequent immunizations were done at monthly intervals in 500 μl ofPBS. Injections were delivered intraperitoneally (i.p.).

PBS injections followed the same schedule and mice were injected with a1:1 mix of PBS/Adjuvant at 400 μl per mouse, or 500 μl of PBS per mouse.SAP injections likewise followed the same schedule using a dose of 100μg per injection.

4. Titration of Mouse Bleeds, Tissue Preparation andImmunohistochemistry

The above methods are described infra in General Materials and Methods.

B. Results

PDAPP mice were injected with either aggregated Aβ42 (aggregated Aβ42),SAP peptides, or phosphate buffered saline. A group of PDAPP mice werealso left as uninjected, positive controls. The titers of the mice toaggregated Aβ42 were monitored every other month from the fourth boostuntil the mice were one year of age. Mice were sacrificed at 13 months.At all time points examined, eight of the nine aggregated Aβ42 micedeveloped a high antibody titer, which remained high throughout theseries of injections (titers greater than 1/10000). The ninth mouse hada low, but measurable titer of approximately 1/1000 (FIG. 1, Table 3).SAPP-injected mice had titers of 1:1,000 to 1:30,000 for this immunogenwith only a single mouse exceeding 1:10,0000.

TABLE 3A Titers at 50% Maximal O.D. Aggregated Aβ injected Mice Age ofPDAPP (months) #100 #101 #102 #103 #104 #105 #106 #107 #108 4 70000150000 15000 120000 1000 15000 50000 60000 100000 6 15000 65000 3000055000 300 15000 15000 50000 60000 8 20000 55000 50000 50000 400 1500018000 50000 60000 10 40000 20000 60000 50000 900 15000 50000 20000 4000012 25000 30000 60000 40000 2700 20000 70000 25000 20000

TABLE 3B Titers at 50% Maximal O.D. PBS injected Mice on both Immunogensat 1/00 Age of PDAPP (months) #113 #114 #115 #116 #117  6 <4 × bkg <4 ×bkg <4 × bkg <4 × bkg <4 × bkg 10   5 × bkg <4 × bkg <4 × bkg <4 × bkg<4 × bkg 12 <4 × bkg <4 × bkg <4 × bkg <4 × bkg <4 × bkg

Sera from PBS-treated mice were titered against aggregated Aβ42 at six,ten and twelve months. At a 1/100 dilution the PBS mice, when titeredagainst aggregated Aβ42, only exceeded 4 times background at one datapoint, otherwise, they were less than 4 times background at all timepoints (Table 3). The SAP-specific response was negligible at these timepoints with all titers less than 300.

Seven out of the nine mice in the aggregated Aβ1-42 treated group had nodetectable amyloid in their brains. In contrast, brain tissue from micein the SAP and PBS groups contained numerous amyloid deposits in thehippocampus, as well as in the frontal and cingulate cortices. Thepattern of deposition was similar to that of untreated controls, withcharacteristic involvement of vulnerable subregions, such as the outermolecular layer of the hippocampal dentate gyrus. One mouse from the Aβ1-42-injected group had a greatly reduced amyloid burden, confined tothe hippocampus. An isolated plaque was identified in another Aβ1-42-treated mouse.

Quantitative image analyses of the amyloid burden in the hippocampusverified the dramatic reduction achieved in the Aβ42(AN1792)-treatedanimals (FIG. 2). The median values of the amyloid burden for the PBSgroup (2.22%), and for the untreated control group (2.65%) weresignificantly greater than for those immunized with AN1792 (0.00%,p=0.0005). In contrast, the median value for the group immunized withSAP peptides (SAPP) was 5.74%. Brain tissue from the untreated, controlmice contained numerous Aβ amyloid deposits visualized with theAβ-specific monoclonal antibody (mAb) 3D6 in the hippocampus, as well asin the retrosplenial cortex. A similar pattern of amyloid deposition wasalso seen in mice immunized with SAPP or PBS (FIG. 2). In addition, inthese latter three groups there was a characteristic involvement ofvulnerable subregions of the brain classically seen in AD, such as theouter molecular layer of the hippocampal dentate gyrus, in all three ofthese groups.

The brains that contained no Aβ deposits were also devoid of neuriticplaques that are typically visualized in PDAPP mice with the human APPantibody 8E5. All of brains from the remaining groups (SAP-injected, PBSand uninjected mice) had numerous neuritic plaques typical of untreatedPDAPP mice. A small number of neuritic plaques were present in one mousetreated with AN1792, and a single cluster of dystrophic neurites wasfound in a second mouse treated with AN1792. Image analyses of thehippocampus, and shown in FIG. 3, demonstrated the virtual eliminationof dystrophic neurites in AN1792-treated mice (median 0.00%) compared tothe PBS recipients (median 0.28%, p=0.0005).

Astrocytosis characteristic of plaque-associated inflammation was alsoabsent in the brains of the Aβ1-42 injected group. The brains from themice in the other groups contained abundant and clustered GFAP-positiveastrocytes typical of Aβ plaque-associated gliosis. A subset of theGFAP-reacted slides were counter-stained with Thioflavin S to localizethe Aβ deposits. The GFAP-positive astrocytes were associated with Aβplaques in the SAP, PBS and untreated controls. No such association wasfound in the plaque-negative Aβ1-42 treated mice, while minimalplaque-associated gliosis was identified in one mouse treated withAN1792.

Image analyses, shown in FIG. 4 for the retrosplenial cortex, verifiedthat the reduction in astrocytosis was significant with a median valueof 1.56% for those treated with AN1792 versus median values greater than6% for groups immunized with SAP peptides, PBS or untreated (p=0.0017)

Evidence from a subset of the Aβ1-42- and PBS-injected mice indicatedplaque-associated MHC II immunoreactivity was absent in the Aβ1-42injected mice, consistent with lack of an Aβ-related inflammatoryresponse.

Sections of the mouse brains were also reacted with a mAb specific witha monoclonal antibody specific for MAC-1, a cell surface protein. MAC-1(CD11b) is an integrin family member and exists as a heterodimer withCD18. The CD11b/CD18 complex is present on monocytes, macrophages,neutrophils and natural killer cells (Mak and Simard). The residentMAC-1-reactive cell type in the brain is likely to be microglia based onsimilar phenotypic morphology in MAC-1 immunoreacted sections.Plaque-associated MAC-1 labeling was lower in the brains of mice treatedwith AN1792 compared to the PBS control group, a finding consistent withthe lack of an Aβ-induced inflammatory response.

C. Conclusion

The lack of Aβ plaques and reactive neuronal and gliotic changes in thebrains of the Aβ1-42-injected mice indicate that no or extremely littleamyloid was deposited in their brains, and pathological consequences,such as gliosis and neuritic pathology, were absent. PDAPP mice treatedwith Aβ1-42 show essentially the same lack of pathology as controlnontransgenic mice. Therefore, Aβ1-42 injections are highly effective inthe prevention of deposition or clearance of human Aβ from brain tissue,and elimination of subsequent neuronal and inflammatory degenerativechanges. Thus, administration of Aβ peptide can have both preventativeand therapeutic benefit in prevention of AD.

II. Dose Response Study

Groups of five-week old, female Swiss Webster mice (N=6 per group) wereimmunized with 300, 100, 33, 11, 3.7, 1.2, 0.4, or 0.13 ug of Aβformulated in CFA/IFA administered intraperitoneally. Three doses weregiven at biweekly intervals followed by a fourth dose one month later.The first dose was emulsified with CFA and the remaining doses wereemulsified with IFA. Animals were bled 4-7 days following eachimmunization starting after the second dose for measurement of antibodytiters. Animals in a subset of three groups, those immunized with 11,33, or 300 μg of antigen, were additionally bled at approximatelymonthly intervals for four months following the fourth immunization tomonitor the decay of the antibody response across a range of doses ofimmunogenic formulations. These animals received a final fifthimmunization at seven months after study initiation. They weresacrificed one week later to measure antibody responses to AN1792 and toperform toxicological analyses.

A declining dose response was observed from 300 to 3.7 μg with noresponse at the two lowest doses. Mean antibody titers are about 1:1000after 3 doses and about 1:10,000 after 4 doses of 11-300 μg of antigen(see FIG. 5).

Antibody titers rose dramatically for all but the lowest dose groupfollowing the third immunization with increases in GMTs ranging from 5-to 25-fold. Low antibody responses were then detectable for even the 0.4μg recipients. The 1.2 and 3.7 μg groups had comparable titers with GMTsof about 1000 and the highest four doses clustered together with GMTs ofabout 25,000, with the exception of the 33 μg dose group with a lowerGMT of 3000. Following the fourth immunization, the titer increase wasmore modest for most groups. There was a clear dose response across thelower antigen dose groups from 0.14 μg to 11 μg ranging from nodetectable antibody for recipients of 0.14 μg to a GMT of 36,000 forrecipients of 11 μg. Again, titers for the four highest dose groups of11 to 300 μg clustered together. Thus following two immunizations, theantibody titer was dependent on the antigen dose across the broad rangefrom 0.4 to 300 μg. By the third immunization, titers of the highestfour doses were all comparable and they remained at a plateau after anadditional immunization.

One month following the fourth immunization, titers were 2- to 3-foldhigher in the 300 μg group than those measured from blood drawn fivedays following the immunization (FIG. 6). This observation suggests thatthe peak anamnestic antibody response occurred later than 5 dayspost-immunization. A more modest (50%) increase was seen at this time inthe 33 μg group. In the 300 μg dose group at two months following thelast dose, GMTs declined steeply by about 70%. After another month, thedecline was less steep at 45% (100 μg) and about 14% for the 33 and 11μg doses. Thus, the rate of decline in circulating antibody titersfollowing cessation of immunization appears to be biphasic with a steepdecline the first month following peak response followed by a moremodest rate of decrease thereafter.

The antibody titers and the kinetics of the response of these SwissWebster mice are similar to those of young heterozygous PDAPP transgenicmice immunized in a parallel manner. Dosages effective to induce animmune response in humans are typically similar to dosages effective inmice.

III. Screen for Therapeutic Efficacy Against Established AD

This assay is designed to test immunogenic agents for activity inarresting or reversing neuropathologic characteristics of AD in agedanimals. Immunizations with 42 amino acid long Aβ (AN1792) were begun ata time point when amyloid plaques are already present in the brains ofthe PDAPP mice.

Over the time course used in this study, untreated PDAPP mice develop anumber of neurodegenerative changes that resemble those found in AD(Games et al., supra and Johnson-Wood et al., Proc. Natl. Acad. Sci. USA94, 1550-1555 (1997)). The deposition of Aβ into amyloid plaques isassociated with a degenerative neuronal response consisting of aberrantaxonal and dendritic elements, called dystrophic neurites. Amyloiddeposits that are surrounded by and contain dystrophic neurites calledneuritic plaques. In both AD and the PDAPP mouse, dystrophic neuriteshave a distinctive globular structure, are immunoreactive with a panelof antibodies recognizing APP and cytoskeletal components, and displaycomplex subcellular degenerative changes at the ultrastructural level.These characteristics allow for disease-relevant, selective andreproducible measurements of neuritic plaque formation in the PDAPPbrains. The dystrophic neuronal component of PDAPP neuritic plaques iseasily visualized with an antibody specific for human APP (monoclonalantibody 8E5), and is readily measurable by computer-assisted imageanalysis. Therefore, in addition to measuring the effects of AN1792 onamyloid plaque formation, we monitored the effects of this treatment onthe development of neuritic dystrophy.

Astrocytes and microglia are non-neuronal cells that respond to andreflect the degree of neuronal injury. GFAP-positive astrocytes and MHCII-positive microglia are commonly observed in AD, and their activationincreases with the severity of the disease. Therefore, we also monitoredthe development of reactive astrocytosis and microgliosis in theAN1792-treated mice.

A. Materials and Methods

Forty-eight, heterozygous female PDAPP mice, 11 to 11.5 months of age,obtained from Charles River, were randomly divided into two groups: 24mice to be immunized with 100 μg of AN1792 and 24 mice to be immunizedwith PBS, each combined with Freund's adjuvant. The AN1792 and PBSgroups were again divided when they reached ˜15 months of age. At 15months of age approximately half of each group of the AN1792- andPBS-treated animals were euthanized (n=10 and 9, respectively), theremainder continued to receive immunizations until termination at ˜18months (n=9 and 12, respectively). A total of 8 animals (5 AN1792, 3PBS) died during the study. In addition to the immunized animals,one-year old (n=10), 15-month old (n=10) and 18-month old (n=10)untreated PDAPP mice were included for comparison in the ELISAs tomeasure Aβ and APP levels in the brain; the one-year old animals werealso included in the immunohistochemical analyses.

Methodology was as in Example I, unless otherwise indicated. US Peptideslot 12 and California Peptides lot ME0339 of AN1792 were used to preparethe antigen for the six immunizations administered prior to the 15-monthtime point. California Peptides lots ME0339 and ME0439 were used for thethree additional immunizations administered between 15 and 18 months.

For immunizations, 100 μg of AN1792 in 200 μl PBS or PBS alone wasemulsified 1:1 (vol:vol) with Complete Freund's adjuvant (CFA) orIncomplete Freund's adjuvant (IFA) or PBS in a final volume of 400 μl.The first immunization was delivered with CFA as adjuvant, the next fourdoses were given with IFA and the final four doses with PBS alonewithout added adjuvant. A total of nine immunizations were given overthe seven-month period on a two-week schedule for the first three dosesfollowed by a four-week interval for the remaining injections. Thefour-month treatment group, euthanized at 15 months of age, receivedonly the first 6 immunizations.

B. Results

1. Effects of Aβ42 (AN1792) Treatment on Amyloid Burden

The results of AN1792 treatment on cortical amyloid burden determined byquantitative image analysis are shown in FIG. 7. The median value ofcortical amyloid burden was 0.28% in a group of untreated 12-month oldPDAPP mice, a value representative of the plaque load in mice at thestudy's initiation. At 18 months, the amyloid burden increased over17-fold to 4.87% in PBS-treated mice, while AN1792-treated mice had agreatly reduced amyloid burden of only 0.01%, notably less than the12-month untreated and both the 15- and 18-month PBS-treated groups. Theamyloid burden was significantly reduced in the AN1792 recipients atboth 15 (96% reduction; p=0.003) and 18 (>99% reduction; p=0.0002)months.

Typically, cortical amyloid deposition in PDAPP mice initiates in thefrontal and retrosplenial cortices (RSC) and progresses in aventral-lateral direction to involve the temporal and entorhinalcortices (EC). Little or no amyloid was found in the EC of 12 month-oldmice, the approximate age at which AN1792 was first administered. After4 months of AN1792 treatment, amyloid deposition was greatly diminishedin the RSC, and the progressive involvement of the EC was entirelyeliminated by AN1792 treatment. The latter observation showed thatAN1792 completely halted the progression of amyloid that would normallyinvade the temporal and ventral cortices, as well as arrested orpossibly reversed deposition in the RSC.

The profound effects of AN1792 treatment on developing cortical amyloidburden in the PDAPP mice are further demonstrated by the 18-month group,which had been treated for seven months. A near complete absence ofcortical amyloid was found in the AN1792-treated mouse, with a totallack of diffuse plaques, as well as a reduction in compacted deposits.

2. Aβ42 (AN1792) Treatment-Associated Cellular and Morphological Changes

A population of Aβ-positive cells was found in brain regions thattypically contain amyloid deposits. Remarkably, in several brains fromAN1792 recipients, very few or no extracellular cortical amyloid plaqueswere found. Most of the Aβ immunoreactivity appeared to be containedwithin cells with large lobular or clumped soma. Phenotypically, thesecells resembled activated microglia or monocytes. They wereimmunoreactive with antibodies recognizing ligands expressed byactivated monocytes and microglia (MHC II and CD11b) and wereoccasionally associated with the wall or lumen of blood vessels.Comparison of near-adjacent sections labeled with Aβ and MHC II-specificantibodies revealed that similar patterns of these cells were recognizedby both classes of antibodies. Detailed examination of theAN1792-treated brains revealed that the MHC II-positive cells wererestricted to the vicinity of the limited amyloid remaining in theseanimals. Under the fixation conditions employed, the cells were notimmunoreactive with antibodies that recognize T cell (CD3, CD3e) or Bcell (CD45RA, CD45RB) ligands or leukocyte common antigen (CD45), butwere reactive with an antibody recognizing leukosialin (CD43) whichcross-reacts with monocytes. No such cells were found in any of thePBS-treated mice.

PDAPP mice invariably develop heavy amyloid deposition in the outermolecular layer of the hippocampal dentate gyrus. The deposition forms adistinct streak within the perforant pathway, a subregion thatclassically contains amyloid plaques in AD. The characteristicappearance of these deposits in PBS-treated mice resembled thatpreviously characterized in untreated PDAPP mice. The amyloid depositionconsisted of both diffuse and compacted plaques in a continuous band. Incontrast, in a number of brains from AN1792-treated mice this patternwas drastically altered. The hippocampal amyloid deposition no longercontained diffuse amyloid, and the banded pattern was completelydisrupted. Instead, a number of unusual punctate structures were presentthat are reactive with anti-Aβ antibodies, several of which appeared tobe amyloid-containing cells.

MHC II-positive cells were frequently observed in the vicinity ofextracellular amyloid in AN1792-treated animals. The pattern ofassociation of Aβ-positive cells with amyloid was very similar inseveral brains from AN1792-treated mice. The distribution of thesemonocytic cells was restricted to the proximity of the deposited amyloidand was entirely absent from other brain regions devoid of Aβ plaques.

Quantitative image analysis of MHC II and MAC I-labeled sectionsrevealed a trend towards increased immunoreactivity in the RSC andhippocampus of AN1792-treated mice compared to the PBS group whichreached significance with the measure of MAC 1 reactivity inhippocampus.

These results are indicative of active, cell-mediated removal of amyloidin plaque-bearing brain regions.

3. AN1792 Effects on Aβ Levels: ELISA Determinations

(a) Cortical Levels

In untreated PDAPP mice, the median level of total Aβ in the cortex at12 months was 1,600 ng/g, which increased to 8,700 ng/g by 15 months(Table 4). At 18 months the value was 22,000 ng/g, an increase of over10-fold during the time course of the experiment. PBS-treated animalshad 8,600 ng/g total Aβ at 15 months which increased to 19,000 ng/g at18 months. In contrast, AN1792-treated animals had 81% less total Aβ at15 months (1,600 ng/g) than the PBS-immunized group. Significantly less(p=0.0001) total Aβ (5,200 ng/g) was found at 18 months when the AN1792and PBS groups were compared (Table 4), representing a 72% reduction inthe Aβ that would otherwise be present. Similar results were obtainedwhen cortical levels of Aβ42 were compared, namely that theAN1792-treated group contained much less Aβ42, but in this case thedifferences between the AN1792 and PBS groups were significant at both15 months (p=0.04) and 18 months (p=0.0001, Table 4).

TABLE 4 Median Aβ Levels (ng/g) in Cortex UNTREATED PBS AN1792 Age TotalAβ42 (n) Total Aβ42 (n) Total Aβ42 (n) 12 1,600 1,300 (10) 15 8,7008,300 (10) 8,600 7,200  (9) 1,600 1,300* (10) 18 22,200 18,500 (10)19,000 15,900 (12) 5,200** 4,000**  (9) *p = 0.0412 **p = 0.0001

(b) Hippocampal Levels

In untreated PDAPP mice, median hippocampal levels of total Aβ at twelvemonths of age were 15,000 ng/g which increased to 51,000 ng/g at 15months and further to 81,000 ng/g at 18 months (Table 5). Similarly, PBSimmunized mice showed values of 40,000 ng/g and 65,000 ng/g at 15 monthsand 18 months, respectively. AN1792 immunized animals exhibited lesstotal Aβ, specifically 25,000 ng/g and 51,000 ng/g at the respective15-month and 18-month timepoints. The 18-month AN1792-treated groupvalue was significantly lower than that of the PBS treated group(p=0.0105; Table 5). Measurement of Aβ42 gave the same pattern ofresults, namely that levels in the AN1792-treated group weresignificantly lower than in the PBS group (39,000 ng/g vs. 57,000 ng/g,respectively; p=0.002) at the 18-month evaluation (Table 3).

TABLE 5 Median Aβ Levels (ng/g) in Hippocampus UNTREATED PBS AN1792 AgeTotal Aβ42 (n) Total Aβ42 (n) Total Aβ42 (n) 12 15,500 11,100 (10) 1551,500 44,400 (10) 40,100 35,70  (9) 24,50 22,100 (10) 18 80,800 64,200(10) 65,400 57,10 (12) 50,90 38,900**  (9) *p = 0.0105 **p = 0.0022

(c) Cerebellar Levels

In 12-month untreated PDAPP mice, the median cerebellar level of totalAβ was 15 ng/g (Table 6). At 15 months, this median increased to 28 ng/gand by 18 months had risen to 35 ng/g. PBS-treated animals displayedmedian total Aβ values of 21 ng/g at 15 months and 43 ng/g at 18 months.AN1792-treated animals were found to have 22 ng/g total Aβ at 15 monthsand significantly less (p=0.002) total Aβ at 18 months (25 ng/g) thanthe corresponding PBS group (Table 6).

TABLE 6 Median Aβ Levels (ng/g) in Cerebellum UNTREATED PBS AN1792 AgeTotal Aβ (n) Total Aβ (n) Total Aβ (n) 12 15.6 (10) 15 27.7 (10) 20.8 (9) 21.7 (10) 18 35.0 (10) 43.1 (12) 24.8*  (9) *p = 0.0018

4. Effects of AN1792 Treatment on APP Levels

APP-α and the full-length APP molecule both contain all or part of theAβ sequence and thus could be potentially impacted by the generation ofan AN1792-directed immune response. In studies to date, a slightincrease in APP levels has been noted as neuropathology increases in thePDAPP mouse. In the cortex, levels of either APP-α/FL (full length) orAPP-α were essentially unchanged by treatment with the exception thatAPP-α was reduced by 19% at the 18-month timepoint in the AN1792-treatedvs. the PBS-treated group. The 18-month AN1792-treated APP values werenot significantly different from values of the 12-month and 15-monthuntreated and 15-month PBS groups. In all cases the APP values remainedwithin the ranges that are normally found in PDAPP mice.

5. Effects of AN1792 Treatment on Neurodegenerative and GlioticPathology

Neuritic plaque burden was significantly reduced in the frontal cortexof AN1792-treated mice compared to the PBS group at both 15 (84%;p=0.03) and 18 (55%; p=0.01) months of age (FIG. 8). The median value ofthe neuritic plaque burden increased from 0.32% to 0.49% in the PBSgroup between 15 and 18 months of age. This contrasted with the greatlyreduced development of neuritic plaques in the AN1792 group, with medianneuritic plaque burden values of 0.05% and 0.22%, in the 15 and 18 monthgroups, respectively.

Immunizations with AN1792 seemed well tolerated and reactiveastrocytosis was also significantly reduced in the RSC of AN1792-treatedmice when compared to the PBS group at both 15 (56%; p=0.011) and 18(39%; p=0.028) months of age (FIG. 9). Median values of the percent ofastrocytosis in the PBS group increased between 15 and 18 months from4.26% to 5.21%. AN1792-treatment suppressed the development ofastrocytosis at both time points to 1.89% and 3.2%, respectively. Thissuggests the neuropil was not being damaged by the clearance process.

6. Antibody Responses

As described above, eleven-month old, heterozygous PDAPP mice (N=24)received a series of 5 immunizations of 100 μg of AN1792 emulsified withFreund's adjuvant and administered intraperitoneally at weeks 0, 2, 4,8, and 12, and a sixth immunization with PBS alone (no Freund'sadjuvant) at week 16. As a negative control, a parallel set of 24age-matched transgenic mice received immunizations of PBS emulsifiedwith the same adjuvants and delivered on the same schedule. Animals werebled within three to seven days following each immunization startingafter the second dose. Antibody responses to AN1792 were measured byELISA. Geometric mean titers (GMT) for the animals that were immunizedwith AN1792 were approximately 1,900, 7,600, and 45,000 following thesecond, third and last (sixth) doses respectively. No Aβ-specificantibody was measured in control animals following the sixthimmunization.

Approximately one-half of the animals were treated for an additionalthree months, receiving immunizations at about 20, 24 and 27 weeks. Eachof these doses was delivered in PBS vehicle alone without Freund'sadjuvant. Mean antibody titers remained unchanged over this time period.In fact, antibody titers appeared to remain stable from the fourth tothe eighth bleed corresponding to a period covering the fifth to theninth injections.

To determine if the Aβ-specific antibodies elicited by immunization thatwere detected in the sera of AN1792-treated mice were also associatedwith deposited brain amyloid, a subset of sections from the AN1792- andPBS-treated mice were reacted with an antibody specific for mouse IgG.In contrast to the PBS group, Aβ plaques in AN1792-treated brains werecoated with endogenous IgG. This difference between the two groups wasseen in both 15- and 18-month groups. Particularly striking was the lackof labeling in the PBS group, despite the presence of a heavy amyloidburden in these mice. These results show that immunization with asynthetic Aβ protein generates antibodies that recognize and bind invivo to the Aβ in amyloid plaques.

7. Cellular-Mediated Immune Responses

Spleens were removed from nine AN1792-immunized and 12 PBS-immunized18-month old PDAPP mice 7 days after the ninth immunization. Splenocyteswere isolated and cultured for 72 h in the presence of Aβ40, Aβ42, orAβ40-1 (reverse order protein). The mitogen Con A served as a positivecontrol. Optimum responses were obtained with >1.7 μM protein. Cellsfrom all nine AN1792-treated animals proliferated in response to eitherAβ1-40 or Aβ1-42 protein, with equal levels of incorporation for bothproteins (FIG. 10, Upper Panel). There was no response to the Aβ40-1reverse protein. Cells from control animals did not respond to any ofthe Aβ proteins (FIG. 10, Lower Panel).

C. Conclusion

The results of this study show that AN1792 immunization of PDAPP micepossessing existing amyloid deposits slows and prevents progressiveamyloid deposition and retard consequential neuropathologic changes inthe aged PDAPP mouse brain. Immunizations with AN1792 essentially haltedamyloid developing in structures that would normally succumb toamyloidosis. Thus, administration of Aβ peptide has therapeutic benefitin the treatment of AD.

IV. Screen of Aβ Fragments

100 PDAPP mice age 9-11 months were immunized with 9 different regionsof APP and Aβ to determine which epitopes convey the efficaciousresponse. The 9 different immunogens and one control are injected i.p.as described above. The immunogens include four human Aβ peptideconjugates 1-12, 13-28, 32-42, 1-5, all coupled to sheep anti-mouse IgGvia a cystine link; an APP polypeptide amino acids 592-695, aggregatedhuman Aβ 1-40, and aggregated human Aβ 25-35, and aggregated rodentAβ42. Aggregated Aβ42 and PBS were used as positive and negativecontrols, respectively. Ten mice were used per treatment group. Titerswere monitored as above and mice were euthanized at the end of 4 monthsof injections. Histochemistry, Aβ levels, and toxicology analysis wasdetermined post mortem.

A. Materials and Methods

1. Preparation of Immunogens

Preparation of coupled Aβ peptides: four human Aβ peptide conjugates(amino acid residues 1-5, 1-12, 13-28, and 33-42, each conjugated tosheep anti-mouse IgG) were prepared by coupling through an artificialcysteine added to the Aβ peptide using the crosslinking reagentsulfo-EMCS. The Aβ peptide derivatives were synthesized with thefollowing final amino acid sequences. In each case, the location of theinserted cysteine residue is indicated by underlining. The Aβ13-28peptide derivative also had two glycine residues added prior to thecarboxyl terminal cysteine as indicated.

Aβ1-12 peptide (SEQ ID NO: 30) NH2-DAEFRHDSGYEVC-COOH Aβ1-5 peptide (SEQID NO: 31) NH2-DAEFRC-COOH Aβ33-42 peptide (SEQ ID NO: 32)NH2-C-amino-heptanoic acid-GLMVGGVVIA-COOH Aβ13-28 peptide (SEQ ID NO:33) Ac-NH-HHQKLVFFAEDVGSNKGGC-COOH

To prepare for the coupling reaction, ten mg of sheep anti-mouse IgG(Jackson ImmunoResearch Laboratories) was dialyzed overnight against 10mM sodium borate buffer, pH 8.5. The dialyzed antibody was thenconcentrated to a volume of 2 mL using an Amicon Centriprep tube. Ten mgsulfo-EMCS

[N (γ-maleimidocuproyloxy) succinimide] (Molecular Sciences Co.) wasdissolved in one mL deionized water. A 40-fold molar excess ofsulfo-EMCS was added dropwise with stirring to the sheep anti-mouse IgGand then the solution was stirred for an additional ten min. Theactivated sheep anti-mouse IgG was purified and buffer exchanged bypassage over a 10 mL gel filtration column (Pierce Presto Column,obtained from Pierce Chemicals) equilibrated with 0.1 M NaPO4, 5 mMEDTA, pH 6.5. Antibody containing fractions, identified by absorbance at280 nm, were pooled and diluted to a concentration of approximately 1mg/mL, using 1.4 mg per OD as the extinction coefficient. A 40-foldmolar excess of Aβ peptide was dissolved in 20 mL of 10 mM NaPO4, pH8.0, with the exception of the Aβ33-42 peptide for which 10 mg was firstdissolved in 0.5 mL of DMSO and then diluted to 20 mL with the 10 mMNaPO4 buffer. The peptide solutions were each added to 10 mL ofactivated sheep anti-mouse IgG and rocked at room temperature for 4 hr.The resulting conjugates were concentrated to a final volume of lessthan 10 mL using an Amicon Centriprep tube and then dialyzed against PBSto buffer exchange the buffer and remove free peptide. The conjugateswere passed through 0.22 μm-pore size filters for sterilization and thenaliquoted into fractions of 1 mg and stored frozen at −20° C. Theconcentrations of the conjugates were determined using the BCA proteinassay (Pierce Chemicals) with horse IgG for the standard curve.Conjugation was documented by the molecular weight increase of theconjugated peptides relative to that of the activated sheep anti-mouseIgG. The Aβ 1-5 sheep anti-mouse conjugate was a pool of twoconjugations, the rest were from a single preparation.

2. Preparation of Aggregated Aβ Peptides

Human 1-40 (AN1528; California Peptides Inc., Lot ME0541), human 1-42(AN1792; California Peptides Inc., Lots ME0339 and ME0439), human 25-35,and rodent 1-42 (California Peptides Inc., Lot ME0218) peptides werefreshly solubilized for the preparation of each set of injections fromlyophilized powders that had been stored desiccated at −20° C. For thispurpose, two mg of peptide were added to 0.9 ml of deionized water andthe mixture was vortexed to generate a relatively uniform solution orsuspension. Of the four, AN1528 was the only peptide soluble at thisstep. A 100 μl aliquot of 10×PBS (1×PBS: 0.15 M NaCl, 0.01 M sodiumphosphate, pH 7.5) was then added at which point AN1528 began toprecipitate. The suspension was vortexed again and incubated overnightat 37° C. for use the next day.

Preparation of the pBx6 protein: An expression plasmid encoding pBx6, afusion protein consisting of the 100-amino acid bacteriophage MS-2polymerase N-terminal leader sequence followed by amino acids 592-695 ofAPP (βAPP) was constructed as described by Oltersdorf et al., J. Biol.Chem. 265, 4492-4497 (1990). The plasmid was transfected into E. coliand the protein was expressed after induction of the promoter. Thebacteria were lysed in 8M urea and pBx6 was partially purified bypreparative SDS PAGE. Fractions containing pBx6 were identified byWestern blot using a rabbit anti-pBx6 polyclonal antibody, pooled,concentrated using an Amicon Centriprep tube and dialysed against PBS.The purity of the preparation, estimated by Coomassie Blue stained SDSPAGE, was approximately 5 to 10%.

B. Results and Discussion

1. Study Design

One hundred male and female, nine- to eleven-month old heterozygousPDAPP transgenic mice were obtained from Charles River Laboratory andTaconic Laboratory. The mice were sorted into ten groups to be immunizedwith different regions of Aβ or APP combined with Freund's adjuvant.Animals were distributed to match the gender, age, parentage and sourceof the animals within the groups as closely as possible. The immunogensincluded four Aβ peptides derived from the human sequence, 1-5, 1-12,13-28, and 33-42, each conjugated to sheep anti-mouse IgG; fouraggregated Aβ peptides, human 1-40 (AN1528), human 1-42 (AN1792), human25-35, and rodent 1-42; and a fusion polypeptide, designated as pBx6,containing APP amino acid residues 592-695. A tenth group was immunizedwith PBS combined with adjuvant as a control.

For each immunization, 100 μg of each Aβ peptide in 200 μl PBS or 200 μgof the APP derivative pBx6 in the same volume of PBS or PBS alone wasemulsified 1:1 (vol:vol) with Complete Freund's adjuvant (CFA) in afinal volume of 400 μl for the first immunization, followed by a boostof the same amount of immunogen in Incomplete Freund's adjuvant (IFA)for the subsequent four doses and with PBS for the final dose.Immunizations were delivered intraperitoneally on a biweekly schedulefor the first three doses, then on a monthly schedule thereafter.Animals were bled four to seven days following each immunizationstarting after the second dose for the measurement of antibody titers.Animals were euthanized approximately one week after the final dose.

2. Aβ and APP Levels in the Brain

Following about four months of immunization with the various Aβ peptidesor the APP derivative, brains were removed from saline-perfused animals.One hemisphere was prepared for immunohistochemical analysis and thesecond was used for the quantitation of Aβ and APP levels. To measurethe concentrations of various forms of beta amyloid peptide and amyloidprecursor protein, the hemisphere was dissected and homogenates of thehippocampal, cortical, and cerebellar regions were prepared in 5 Mguanidine. These were diluted and the level of amyloid or APP wasquantitated by comparison to a series of dilutions of standards of Aβpeptide or APP of known concentrations in an ELISA format.

The median concentration of total Aβ for the control group immunizedwith PBS was 5.8-fold higher in the hippocampus than in the cortex(median of 24,318 ng/g hippocampal tissue compared to 4,221 ng/g for thecortex). The median level in the cerebellum of the control group (23.4ng/g tissue) was about 1,000-fold lower than in the hippocampus. Theselevels are similar to those that we have previously reported forheterozygous PDAPP transgenic mice of this age (Johnson-Woods et al.,1997, supra).

For the cortex, a subset of treatment groups had median total Aβ andAβ1-42 levels which differed significantly from those of the controlgroup (p<0.05), those animals receiving AN1792, rodent Aβ1-42 or theAβ1-5 peptide conjugate as shown in FIG. 11. The median levels of totalAβ were reduced by 75%, 79% and 61%, respectively, compared to thecontrol for these treatment groups. There were no discernablecorrelations between Aβ-specific antibody titers and Aβ levels in thecortical region of the brain for any of the groups.

In the hippocampus, the median reduction of total Aβ associated withAN1792 treatment (46%, p=0.0543) was not as great as that observed inthe cortex (75%, p=0.0021). However, the magnitude of the reduction wasfar greater in the hippocampus than in the cortex, a net reduction of11,186 ng/g tissue in the hippocampus versus 3,171 ng/g tissue in thecortex. For groups of animals receiving rodent Aβ1-42 or Aβ1-5, themedian total Aβ levels were reduced by 36% and 26%, respectively.However, given the small group sizes and the high variability of theamyloid peptide levels from animal to animal within both groups, thesereductions were not significant. When the levels of Aβ1-42 were measuredin the hippocampus, none of the treatment-induced reductions reachedsignificance. Thus, due to the smaller Aβ burden in the cortex, changesin this region are a more sensitive indicator of treatment effects. Thechanges in Aβ levels measured by ELISA in the cortex are similar, butnot identical, to the results from the immunohistochemical analysis (seebelow).

Total Aβ was also measured in the cerebellum, a region typicallyminimally affected with AD pathology. None of the median Aβconcentrations of any of the groups immunized with the various Aβpeptides or the APP derivative differed from that of the control groupin this region of the brain. This result suggests that non-pathologicallevels of Aβ are unaffected by treatment.

APP concentration was also determined by ELISA in the cortex andcerebellum from treated and control mice. Two different APP assays wereutilized. The first, designated APP-α/FL, recognizes both APP-alpha (α,the secreted form of APP which has been cleaved within the Aβ sequence),and full-length forms (FL) of APP, while the second recognizes onlyAPP-α. In contrast to the treatment-associated diminution of Aβ in asubset of treatment groups, the levels of APP were unchanged in all ofthe treated compared to the control animals. These results indicate thatthe immunizations with Aβ peptides are not depleting APP; rather thetreatment effect is specific to Aβ.

In summary, total Aβ and Aβ1-42 levels were significantly reduced in thecortex by treatment with AN1792, rodent Aβ1-42 or Aβ1-5 conjugate. Inthe hippocampus, total Aβ was significantly reduced only by AN1792treatment. No other treatment-associated changes in Aβ or APP levels inthe hippocampal, cortical or cerebellar regions were significant.

2. Histochemical Analyses

Brains from a subset of six groups were prepared for immunohistochemicalanalysis, three groups immunized with the Aβ peptide conjugates Aβ1-5,Aβ1-12, and Aβ13-28; two groups immunized with the full length Aβaggregates AN1792 and AN1528 and the PBS-treated control group. Theresults of image analyses of the amyloid burden in brain sections fromthese groups are shown in FIG. 12. There were significant reductions ofamyloid burden in the cortical regions of three of the treatment groupsversus control animals. The greatest reduction of amyloid burden wasobserved in the group receiving AN1792 where the mean value was reducedby 97% (p=0.001). Significant reductions were also observed for thoseanimals treated with AN1528 (95%, p=0.005) and the Aβ1-5 peptideconjugate (67%, p=0.02).

The results obtained by quantitation of total Aβ or Aβ1-42 by ELISA andamyloid burden by image analysis differ to some extent. Treatment withAN1528 had a significant impact on the level of cortical amyloid burdenwhen measured by quantitative image analysis but not on theconcentration of total Aβ in the same region when measured by ELISA. Thedifference between these two results is likely to be due to thespecificities of the assays. Image analysis measures only insoluble Aβaggregated into plaques. In contrast, the ELISA measures all forms ofAβ, both soluble and insoluble, monomeric and aggregated. Since thedisease pathology is thought to be associated with the insolubleplaque-associated form of Aβ, the image analysis technique may have moresensitivity to reveal treatment effects. However since the ELISA is amore rapid and easier assay, it is very useful for screening purposes.Moreover it may reveal that the treatment-associated reduction of Aβ isgreater for plaque-associated than total Aβ.

To determine if the Aβ-specific antibodies elicited by immunization inthe treated animals reacted with deposited brain amyloid, a subset ofthe sections from the treated animals and the control mice were reactedwith an antibody specific for mouse IgG. In contrast to the PBS group,Aβ-containing plaques were coated with endogenous IgG for animalsimmunized with the Aβ peptide conjugates Aβ1-5, Aβ1-12, and Aβ13-28; andthe full length Aβ aggregates AN1792 and AN1528. Brains from animalsimmunized with the other Aβ peptides or the APP peptide pBx6 were notanalyzed by this assay.

3. Measurement of Antibody Titers

Mice were bled four to seven days following each immunization startingafter the second immunization, for a total of five bleeds. Antibodytiters were measured as Aβ1-42-binding antibody using a sandwich ELISAwith plastic multi-well plates coated with Aβ1-42. As shown in FIG. 13,peak antibody titers were elicited following the fourth dose for thosefour immunization formulations which elicited the highest titers ofAN1792-specific antibodies: AN1792 (peak GMT: 94,647), AN1528 (peak GMT:88,231), Aβ1-12 conjugate (peak GMT: 47,216) and rodent Aβ1-42 (peakGMT: 10,766). Titers for these groups declined somewhat following thefifth and sixth doses. For the remaining five immunogens, peak titerswere reached following the fifth or the sixth dose and these were ofmuch lower magnitude than those of the four highest titer groups: Aβ1-5conjugate (peak GMT: 2,356), pBx6 (peak GMT: 1,986), Aβ13-28 conjugate(peak GMT: 1,183), Aβ33-42 conjugate (peak GMT: 658), Aβ25-35 (peak GMT:125). Antibody titers were also measured against the homologous peptidesusing the same ELISA sandwich format for a subset of the immunogens,those groups immunized with Aβ1-5, Aβ13-28, Aβ25-35, Aβ33-42 or rodentAβ1-42. These titers were about the same as those measured againstAβ1-42 except for the rodent Aβ1-42 immunogen in which case antibodytiters against the homologous immunogen were about two-fold higher. Themagnitude of the AN1792-specific antibody titer of individual animals orthe mean values of treatment groups did not correlate with efficacymeasured as the reduction of Aβ in the cortex.

4. Lymphoproliferative Responses

Aβ-dependent lymphoproliferation was measured using spleen cellsharvested approximately one week following the final, sixth,immunization. Freshly harvested cells, 105 per well, were cultured for 5days in the presence of Aβ1-40 at a concentration of 5 μM forstimulation. Cells from a subset of seven of the ten groups were alsocultured in the presence of the reverse peptide, Aβ40-1. As a positivecontrol, additional cells were cultured with the T cell mitogen, PHA,and, as a negative control, cells were cultured without added peptide.

Lymphocytes from a majority of the animals proliferated in response toPHA. There were no significant responses to the Aβ40-1 reverse peptide.Cells from animals immunized with the larger aggregated Aβ peptides,AN1792, rodent Aβ1-42 and AN1528 proliferated robustly when stimulatedwith Aβ1-40 with the highest cpm in the recipients of AN1792. One animalin each of the groups immunized with Aβ1-12 conjugate, Aβ13-28 conjugateand Aβ25-35 proliferated in response to Aβ1-40. The remaining groupsreceiving Aβ1-5 conjugate, Aβ33-42 conjugate pBx6 or PBS had no animalswith an Aβ-stimulated response. These results are summarized in Table 7below.

TABLE 7 Immunogen Conjugate Aβ Amino Acids Responders Aβ1-5 Yes  5-mer0/7 Aβ1-12 Yes 12-mer 1/8 Aβ13-28 Yes 16-mer 1/9 Aβ25-35 11-mer 1/9Aβ33-42 Yes 10-mer  0/10 Aβ1-40 40-mer 5/8 Aβ1-42 42-mer 9/9 r Aβ1-4242-mer 8/8 pBx6 0/8 PBS  0-mer 0/8

These results show that AN1792 and AN1528 stimulate strong T cellresponses, most likely of the CD4+ phenotype. The absence of anAβ-specific T cell response in animals immunized with Aβ1-5 is notsurprising since peptide epitopes recognized by CD4+ T cells are usuallyabout 15 amino acids in length, although shorter peptides can sometimesfunction with less efficiency. Thus the majority of helper T cellepitopes for the four conjugate peptides are likely to reside in the IgGconjugate partner, not in the Aβ region. This hypothesis is supported bythe very low incidence of proliferative responses for animals in each ofthese treatment groups. Since the Aβ1-5 conjugate was effective atsignificantly reducing the level of Aβ in the brain, in the apparentabsence of Aβ-specific T cells, the key effector immune response inducedby immunization with this peptide appears to be antibody.

Lack of T-cell and low antibody response from fusion peptide pBx6,encompassing APP amino acids 592-695 including all of the Aβ residuesmay be due to the poor immunogenicity of this particular preparation.The poor immunogenicity of the Aβ25-35 aggregate is likely due to thepeptide being too small to be likely to contain a good T cell epitope tohelp the induction of an antibody response. It is anticipated thatconjugation of this peptide to a carrier protein would render it moreimmunogenic.

V. Preparation of Polyclonal Antibodies for Passive Protection

125 non-transgenic mice were immunized with Aβ, plus adjuvant, andeuthanized at 4-5 months. Blood was collected from immunized mice. IgGwas separated from other blood components. Antibody specific for theimmunogen may be partially purified by affinity chromatography. Anaverage of about 0.5-1 mg of immunogen-specific antibody is obtained permouse, giving a total of 60-120 mg.

VI. Passive Immunization with Antibodies to Aβ

Groups of 7-9 month old PDAPP mice each were injected with 0.5 mg in PBSof polyclonal anti-Aβ or specific anti-Aβ monoclonals as shown below.All antibody preparations were purified to have low endotoxin levels.Monoclonals can be prepared against a fragment by injecting the fragmentor longer form of Aβ into a mouse, preparing hybridomas and screeningthe hybridomas for an antibody that specifically binds to a desiredfragment of Aβ without binding to other nonoverlapping fragments of Aβ.

TABLE 8 Antibody Epitope 2H3 Aβ 1-12 10D5 Aβ 1-12 266 Aβ 13-28 21F12 Aβ33-42 Mouse polyclonal Anti-Aggregated Aβ42 anti-human Aβ42

Mice were injected ip as needed over a 4 month period to maintain acirculating antibody concentration measured by ELISA titer of greaterthan 1/1000 defined by ELISA to Aβ42 or other immunogen. Titers weremonitored as above and mice were euthanized at the end of 6 months ofinjections. Histochemistry, Aβ levels and toxicology were performed postmortem. Ten mice were used per group. Additional studies of passiveimmunization are described in Examples XI and XII below.

VII. Comparison of Different Adjuvants

This example compares CFA, alum, an oil-in water emulsion and MPL forcapacity to stimulate an immune response.

A. Materials and Methods

1. Study Design

One hundred female Hartley strain six-week old guinea pigs, obtainedfrom Elm Hill Breeding Laboratories, Chelmsford, Mass., were sorted intoten groups to be immunized with AN1792 or a palmitoylated derivativethereof combined with various adjuvants. Seven groups receivedinjections of AN1792 (33 μg unless otherwise specified) combined with a)PBS, b) Freund's adjuvant, c) MPL, d) squalene, e) MPL/squalene f) lowdose alum, or g) high dose alum (300 μg AN1792). Two groups receivedinjections of a palmitoylated derivative of AN1792 (33 μg) combined witha) PBS or b) squalene. A final, tenth group received PBS alone withoutantigen or additional adjuvant. For the group receiving Freund'sadjuvant, the first dose was emulsified with CFA and the remaining fourdoses with IFA. Antigen was administered at a dose of 33 μg for allgroups except the high dose alum group, which received 300 μg of AN1792.Injections were administered intraperitoneally for CFA/IFA andintramuscularly in the hind limb quadriceps alternately on the right andleft side for all other groups. The first three doses were given on abiweekly schedule followed by two doses at a monthly interval). Bloodwas drawn six to seven days following each immunization, starting afterthe second dose, for measurement of antibody titers.

2. Preparation of Immunogens

Two mg Aβ42 (California Peptide, Lot ME0339) was added to 0.9 ml ofdeionized water and the mixture was vortexed to generate a relativelyuniform suspension. A 100 μl aliquot of 10×PBS (1×PBS, 0.15 M NaCl, 0.01M sodium phosphate, pH 7.5) was added. The suspension was vortexed againand incubated overnight at 37° C. for use the next day. Unused Aβ1-42was stored with desiccant as a lyophilized powder at −20° C.

A palmitoylated derivative of AN1792 was prepared by coupling palmiticanhydride, dissolved in dimethyl formamide, to the amino terminalresidue of AN1792 prior to removal of the nascent peptide from the resinby treatment with hydrofluoric acid.

To prepare immunogenic formulation doses with Complete Freund's adjuvant(CFA) (group 2), 33 μg of AN1792 in 200 μl PBS was emulsified 1:1(vol:vol) with CFA in a final volume of 400 μl for the firstimmunization. For subsequent immunizations, the antigen was similarlyemulsified with Incomplete Freund's adjuvant (IFA).

To prepare formulation doses with MPL for groups 5 and 8, lyophilizedpowder (Ribi ImmunoChem Research, Inc., Hamilton, Mont.) was added to0.2% aqueous triethylamine to a final concentration of 1 mg/ml andvortexed. The mixture was heated to 65 to 70° C. for 30 sec to create aslightly opaque uniform suspension of micelles. The solution was freshlyprepared for each set of injections. For each injection in group 5, 33μg of AN1792 in 16.5 μl PBS, 50 μg of MPL (50 μl) and 162 μl of PBS weremixed in a borosilicate tube immediately before use.

To prepare formulation doses with the low oil-in-water emulsion, AN1792in PBS was added to 5% squalene, 0.5% Tween 80, 0.5% Span 85 in PBS toreach a final single dose concentration of 33 μg AN1792 in 250 μl (group6). The mixture was emulsified by passing through a two-chamberedhand-held device 15 to 20 times until the emulsion droplets appeared tobe about equal in diameter to a 1.0 μm diameter standard latex bead whenviewed under a microscope. The resulting suspension was opalescent,milky white. The emulsions were freshly prepared for each series ofinjections. For group 8, MPL in 0.2% triethylamine was added at aconcentration of 50 μg per dose to the squalene and detergent mixturefor emulsification as noted above. For the palmitoyl derivative (group7), 33 μg per dose of palmitoyl-NH-Aβ1-42 was added to squalene andvortexed. Tween 80 and Span 85 were then added with vortexing. Thismixture was added to PBS to reach final concentrations of 5% squalene,0.5% Tween 80, 0.5% Span 85 and the mixture was emulsified as notedabove.

To prepare formulation doses with alum (groups 9 and 10), AN1792 in PBSwas added to Alhydrogel (aluminum hydroxide gel, Accurate, Westbury,N.Y.) to reach concentrations of 33 μg (low dose, group 9) or 300 μg(high dose, group 10) AN1792 per 5 mg of alum in a final dose volume of250 μl. The suspension was gently mixed for 4 hr at RT.

3. Measurement of Antibody Titers

Guinea pigs were bled six to seven days following immunization startingafter the second immunization for a total of four bleeds. Antibodytiters against Aβ42 were measured by ELISA as described in GeneralMaterials and Methods.

4. Tissue Preparation

After about 14 weeks, all guinea pigs were euthanized by administrationof CO₂. Cerebrospinal fluid was collected and the brains were removedand three brain regions (hippocampus, cortex and cerebellum) weredissected and used to measure the concentration of total Aβ proteinusing ELISA.

B. Results

1. Antibody Responses

There was a wide range in the potency of the various adjuvants whenmeasured as the antibody response to AN1792 following immunization. Asshown in FIG. 14, when AN1792 was administered in PBS, no antibody wasdetected following two or three immunizations and negligible responseswere detected following the fourth and fifth doses with geometric meantiters (GMTs) of only about 45. The o/w emulsion induced modest titersfollowing the third dose (GMT 255) that were maintained following thefourth dose (GMT 301) and fell with the final dose (GMT 54). There was aclear antigen dose response for AN1792 bound to alum with 300 μg beingmore immunogenic at all time points than 33 μg. At the peak of theantibody response, following the fourth immunization, the differencebetween the two doses was 43% with GMTs of about 1940 (33 μg) and 3400(300 μg). The antibody response to 33 μg AN1792 plus MPL was verysimilar to that generated with almost a ten-fold higher dose of antigen(300 μg) bound to alum. The addition of MPL to an o/w emulsion decreasedthe potency of the formulation relative to that with MPL as the soleadjuvant by as much as 75%. A palmitoylated derivative of AN1792 wascompletely non-immunogenic when administered in PBS and gave modesttiters when presented in an o/w emulsion with GMTs of 340 and 105 forthe third and fourth bleeds. The highest antibody titers were generatedwith Freund's adjuvant with a peak GMT of about 87,000, a value almost30-fold greater than the GMTs of the next two most potent formulations,MPL and high dose AN1792/alum.

The most promising adjuvants identified in this study are MPL and alum.Of these two, MPL appears preferable because a 10-fold lower antigendose was required to generate the same antibody response as obtainedwith alum. The response can be increased by increasing the dose ofantigen and/or adjuvant and by optimizing the immunization schedule. Theo/w emulsion was a very weak adjuvant for AN1792 and adding an o/wemulsion to MPL adjuvant diminished the intrinsic adjuvant activity ofMPL alone.

2. Aβ Levels in the Brain

At about 14 weeks the guinea pigs were deeply anesthetized, thecerebrospinal fluid (CSF) was drawn and brains were excised from animalsin a subset of the groups, those immunized with Freund's adjuvant (group2), MPL (group 5), alum with a high dose, 300 μg, of AN1792 (group 10)and the PBS immunized control group (group 3). To measure the level ofAβ peptide, one hemisphere was dissected and homogenates of thehippocampal, cortical, and cerebellar regions were prepared in 5 Mguanidine. These were diluted and quantitated by comparison to a seriesof dilutions of Aβ standard protein of known concentrations in an ELISAformat. The levels of Aβ protein in the hippocampus, the cortex and thecerebellum were very similar for all four groups despite the wide rangeof antibody responses to Aβ elicited by these formulations. Mean Aβlevels of about 25 ng/g tissue were measured in the hippocampus, 21 ng/gin the cortex, and 12 ng/g in the cerebellum. Thus, the presence of ahigh circulating antibody titer to Aβ for almost three months in some ofthese animals did not alter the total Aβ levels in their brains. Thelevels of Aβ in the CSF were also quite similar between the groups. Thelack of large effect of AN1792 immunization on endogenous Aβ indicatesthat the immune response is focused on pathological formations of Aβ.

VIII. Immune Response to Different Adjuvants in Mice

Six-week old female Swiss Webster mice were used for this study with10-13 animals per group. Immunizations were given on days 0, 14, 28, 60,90 and 20 administered subcutaneously in a dose volume of 200 μl. PBSwas used as the buffer for all formulations. Animals were bleed sevendays following each immunization starting after the second dose foranalysis of antibody titers by ELISA. The treatment regime of each groupis summarized in Table 9.

TABLE 9 Experimental Design Dose Group N^(a) Adjuvant^(b) Dose Antigen(μg) 1 10 MPL 12.5 μg AN1792 33 2 10 MPL 25 μg AN1792 33 3 10 MPL 50 μgAN1792 33 4 13 MPL 125 μg AN1792 33 5 13 MPL 50 μg AN1792 150 6 13 MPL50 μg AN1528 33 7 10 PBS AN1792 33 8 10 PBS None 9 10 Squalene 5% AN179233 emulsified 10 10 Squalene 5% AN1792 33 admixed 11 10 Alum 2 mg AN179233 12 13 MPL + Alum 50 μg/2 mg AN1792 33 13 10 QS-21 5 μg AN1792 33 1410 QS-21 10 μg AN1792 33 15 10 QS-21 25 AN1792 AN1792 33 16 13 QS-21 25AN1792 AN1792 150 17 13 QS-21 25 AN1792 AN1528 33 18 13 QS-21 + MPL 25μg/50 μg AN1792 33 19 13 QS-21 + 25 μg/2 mg AN1792 33 Alum Footnotes:^(a)Number of mice in each group at the initiation of the experiment.^(b)The adjuvants are noted. The buffer for all these formulations wasPBS. For group 8, there was no adjuvant and no antigen.The ELISA titers of antibodies against Aβ42 in each group are shown inTable 10 below.

TABLE 10 Geometric Mean Antibody Titers Week of Bleed Treatment Group2.9 5.0 8.7 12.9 16.7 1 248 1797 2577 6180 4177 2 598 3114 3984 52876878 3 1372 5000 7159 12333 12781 4 1278 20791 14368 20097 25631 5 328826242 13229 9315 23742 6 61 2536 2301 1442 4504 7 37 395 484 972 2149 825 25 25 25 25 9 25 183 744 952 1823 10 25 89 311 513 817 11 29 708 26182165 3666 12 198 1458 1079 612 797 13 38 433 566 1080 626 14 104 5413247 1609 838 15 212 2630 2472 1224 1496 16 183 2616 6680 2085 1631 1728 201 375 222 1540 18 31699 15544 23095 6412 9059 19 63 243 554 299 441The table shows that the highest titers were obtained for groups 4, 5and 18, in which the adjuvants were 125 μg MPL, 50 μg MPL and QS-21 plusMPL.

IX. Therapeutic Efficacy of Different Adjuvants

A therapeutic efficacy study was conducted in PDAPP transgenic mice witha set of adjuvants suitable for use in humans to determine their abilityto potentiate immune responses to Aβ and to induce the immune-mediatedclearance of amyloid deposits in the brain.

One hundred eighty male and female, 7.5- to 8.5-month old heterozygousPDAPP transgenic mice were obtained from Charles River Laboratories. Themice were sorted into nine groups containing 15 to 23 animals per groupto be immunized with AN1792 or AN1528 combined with various adjuvants.Animals were distributed to match the gender, age, and parentage of theanimals within the groups as closely as possible. The adjuvants includedalum, MPL, and QS-21, each combined with both antigens, and Freund'sadjuvant (FA) combined with only AN1792. An additional group wasimmunized with AN1792 formulated in PBS buffer plus the preservativethimerosal without adjuvant. A ninth group was immunized with PBS aloneas a negative control.

Preparation of aggregated Aβ peptides: human Aβ1-40 (AN1528; CaliforniaPeptides Inc., Napa, Calif.; Lot ME0541) and human Aβ1-42 (AN1792;California Peptides Inc., Lot ME0439) peptides were freshly solubilizedfor the preparation of each set of injections from lyophilized powdersthat had been stored desiccated at −20° C. For this purpose, two mg ofpeptide were added to 0.9 ml of deionized water and the mixture wasvortexed to generate a relatively uniform solution or suspension. AN1528was soluble at this step, in contrast to AN1792. A 100 μl aliquot of10×PBS (1×PBS: 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) was thenadded at which point AN1528 began to precipitate. The suspensions werevortexed again and incubated overnight at 37° C. for use the next day.

To prepare formulation doses with alum (Groups 1 and 5), Aβ peptide inPBS was added to Alhydrogel (two percent aqueous aluminum hydroxide gel,Sargeant, Inc., Clifton, N.J.) to reach concentrations of 100 μg Aβpeptide per 1 mg of alum. 10×PBS was added to a final dose volume of 200μl in 1×PBS. The suspension was then gently mixed for approximately 4 hrat RT prior to injection.

To prepare formulation doses for with MPL (Groups 2 and 6), lyophilizedpowder (Ribi ImmunoChem Research, Inc., Hamilton, Mont.; Lot67039-E0896B) was added to 0.2% aqueous triethylamine to a finalconcentration of 1 mg/ml and vortexed. The mixture was heated to 65 to70° C. for 30 sec to create a slightly opaque uniform suspension ofmicelles. The solution was stored at 4° C. For each set of injections,100 μg of peptide per dose in 50 μl PBS, 50 μg of MPL per dose (50 μl)and 100 μl of PBS per dose were mixed in a borosilicate tube immediatelybefore use.

To prepare formulation doses with QS-21 (Groups 3 and 7), lyophilizedpowder (Aquila, Framingham, Mass.; Lot A7018R) was added to PBS, pH6.6-6.7 to a final concentration of 1 mg/ml and vortexed. The solutionwas stored at −20° C. For each set of injections, 100 μg of peptide perdose in 50 μl PBS, 25 μg of QS-21 per dose in 25 μl PBS and 125 μl ofPBS per dose were mixed in a borosilicate tube immediately before use.

To prepare formulation doses with Freund's Adjuvant (Group 4), 100 μg ofAN1792 in 200 μl PBS was emulsified 1:1 (vol:vol) with Complete Freund'sAdjuvant (CFA) in a final volume of 400 μl for the first immunization.For subsequent immunizations, the antigen was similarly emulsified withIncomplete Freund's Adjuvant (IFA). For the formulations containing theadjuvants alum, MPL or QS-21, 100 μg per dose of AN1792 or AN1528 wascombined with alum (1 mg per dose) or MPL (50 μg per dose) or QS-21 (25μg per dose) in a final volume of 200 μl PBS and delivered bysubcutaneous inoculation on the back between the shoulder blades. Forthe group receiving FA, 100 μg of AN1792 was emulsified 1:1 (vol:vol)with Complete Freund's adjuvant (CFA) in a final volume of 400 μl anddelivered intraperitoneally for the first immunization, followed by aboost of the same amount of immunogen in Incomplete Freund's adjuvant(IFA) for the subsequent five doses. For the group receiving AN1792without adjuvant, 10 μg AN1792 was combined with 5 μg thimerosal in afinal volume of 50 μl PBS and delivered subcutaneously. The ninth,control group received only 200 μl PBS delivered subcutaneously.Immunizations were given on a biweekly schedule for the first threedoses, then on a monthly schedule thereafter on days 0, 16, 28, 56, 85and 112. Animals were bled six to seven days following each immunizationstarting after the second dose for the measurement of antibody titers.Animals were euthanized approximately one week after the final dose.Outcomes were measured by ELISA assay of Aβ and APP levels in brain andby immunohistochemical evaluation of the presence of amyloid plaques inbrain sections. In addition, Aβ-specific antibody titers, andAβ-dependent proliferative and cytokine responses were determined.

Table 10 shows that the highest antibody titers to Aβ1-42 were elicitedwith FA and AN1792, titers which peaked following the fourthimmunization (peak GMT: 75,386) and then declined by 59% after thefinal, sixth immunization. The peak mean titer elicited by MPL withAN1792 was 62% lower than that generated with FA (peak GMT: 28,867) andwas also reached early in the immunization scheme, after 3 doses,followed by a decline to 28% of the peak value after the sixthimmunization. The peak mean titer generated with QS-21 combined withAN1792 (GMT: 1,511) was about 5-fold lower than obtained with MPL. Inaddition, the kinetics of the response were slower, since an additionalimmunization was required to reach the peak response. Titers generatedby alum-bound AN1792 were marginally greater than those obtained withQS-21 and the response kinetics were more rapid. For AN1792 delivered inPBS with thimerosal the frequency and size of titers were barely greaterthan that for PBS alone. The peak titers generated with MPL and AN1528(peak GMT 3099) were about 9-fold lower than those with AN1792.Alum-bound AN1528 was very poorly immunogenic with low titers generatedin only some of the animals. No antibody responses were observed in thecontrol animals immunized with PBS alone.

TABLE 11 Geometric Mean Antibody Titers^(a) Week of Bleed Treatment 3.35.0 9.0 13.0 17.0 Alum/ 102  1,081  2,366 1,083   572 AN1792 (12/21)^(b) (17/20) (21/21) (19/21) (18/21) MPL/ 6241  28,867  1,12425,665 8,204 AN1792 (21/21) (21/21) (21/21) (20/20) (20/20) QS-21/ 30  227   327 1,511 1,188 AN1792  (1/20) (10/19) (10/19) (17/18) (14/18)CFA/ 10,076    61,279  75,386 41,628  30,574  AN1792 (15/15) (15/15)(15/15) (15/15) (15/15) Alum/ 25   33    39   37   31 AN1528  (0/21) (1/21)  (3/20)  (1/20)  (2/20) MPL/ 184  2,591  1,653 1,156 3,099AN1528 (15/21) (20/21) (21/21) (20/20) (20/20) QS-21/ 29   221    51  820 2,994 AN1528  (1/22) (13/22)  (4/22) (20/22) (21/22) PBS plus 25  33    39   37   47 Thimerosal  (0/16)  (2/16)  (4/16)  (3/16)  (4/16)PBS 25   25    25   25   25  (0/16)  (0/16)  (0/15)  (0/12)  (0/16)Footnotes: ^(a)Geometric mean antibody titers measured against Aβ1-42^(b)Number of responders per group

The results of AN1792 or AN1528 treatment with various adjuvants, orthimerosal on cortical amyloid burden in 12-month old mice determined byELISA are shown in FIG. 15. In PBS control PDAPP mice, the median levelof total Aβ in the cortex at 12 months was 1,817 ng/g. Notably reducedlevels of Aβ were observed in mice treated with AN1792 plus CFA/IFA,AN1792 plus alum, AN1792 plus MPL and QS-21 plus AN1792. The reductionreached statistical significance at the p<0.05 level only for AN1792plus CFA/IFA. However, as shown in Examples I and III, the effects ofimmunization in reducing Aβ levels become substantially greater in 15month and 18 month old mice. Thus, it is anticipated that additionalformulations, particularly AN1792 plus alum, AN1792 plus MPL and AN1792plus QS-21 compositions will provide more positive results in treatmentof older mice. By contrast, the AN1792 plus the preservative thimerosalshowed a median level of Aβ about the same as that in the PBS treatedmice. Similar results were obtained when cortical levels of Aβ42 werecompared. The median level of Aβ42 in PBS controls was 1624 ng/g.Notably reduced median levels of 403, 1149, 620 and 714 were observed inthe mice treated with AN1792 plus CFA/IFA, AN1792 plus alum, AN1792 plusMPL and AN1792 plus QS-21 respectively, with the reduction achievingstatistical significance (p=0.05) for the AN1792 CFA/IFA treatmentgroup. The median level in the AN1792 thimerosal treated mice was 1619ng/g Aβ42.

X. Toxicity Analysis

Tissues were collected for histopathologic examination at thetermination of studies described in Examples II, III and VII. Inaddition, hematology and clinical chemistry were performed on terminalblood samples from Examples III and VI. Most of the major organs wereevaluated, including brain, pulmonary, lymphoid, gastrointestinal,liver, kidney, adrenal and gonads. Although sporadic lesions wereobserved in the study animals, there were no obvious differences, eitherin tissues affected or lesion severity, between AN1792 treated anduntreated animals. There were no unique histopathological lesions notedin AN-1528-immunized animals compared to PBS-treated or untreatedanimals. There were also no differences in the clinical chemistryprofile between adjuvant groups and the PBS treated animals in ExampleVI. Although there were significant increases in several of thehematology parameters between animals treated with AN1792 and Freund'sadjuvant in Example VI relative to PBS treated animals, these type ofeffects are expected from Freund's adjuvant treatment and theaccompanying peritonitis and do not indicate any adverse effects fromAN1792 treatment. Although not part of the toxicological evaluation,PDAPP mouse brain pathology was extensively examined as part of theefficacy endpoints. No sign of treatment related adverse effect on brainmorphology was noted in any of the studies. These results indicate thatAN1792 treatment is well tolerated and at least substantially free ofside effects.

XI. Therapeutic Treatment with Anti-Aβ Antibodies

The experiments described in this section were carried out in order totest the abilities of various monoclonal and polyclonal antibodiesagainst Aβ to inhibit accumulation of Aβ in the brain of heterozygotictransgenic mice.

A. Study 1

1. Study Design

Sixty male and female, heterozygous PDAPP transgenic mice, 8.5 to 10.5months of age were obtained from Charles River Laboratory. The mice weresorted into six groups to be treated with various antibodies directed toAβ. Animals were distributed to match the gender, age, parentage andsource of the animals within the groups as closely as possible. As shownin Table 12, the antibodies included four murine Aβ-specific monoclonalantibodies, 2H3 (directed to Aβ residues 1-12), 10D5 (directed to Aβresidues 1-16), 266 (directed to Aβ residues 13-28 and binds tomonomeric but not to aggregated AN1792), 21F12 (directed to Aβ residues33-42). A fifth group was treated with an Aβ-specific polyclonalantibody fraction (raised by immunization with aggregated AN1792). Thenegative control group received the diluent, PBS, alone withoutantibody.

The monoclonal antibodies were injected at a dose of about 10 mg/kg(assuming that the mice weighed 50 g). Injections were administeredintraperitoneally every seven days on average to maintain anti-Aβ titersabove 1000. Although lower titers were measured for mAb 266 since itdoes not bind well to the aggregated AN1792 used as the capture antigenin the assay, the same dosing schedule was maintained for this group.The group receiving monoclonal antibody 2H3 was discontinued within thefirst three weeks since the antibody was cleared too rapidly in vivo.Animals were bled prior to each dosing for the measurement of antibodytiters. Treatment was continued over a six-month period for a total of196 days. Animals were euthanized one week after the final dose.

TABLE 12 EXPERIMENTAL DESIGN OF STUDY 006 Treatment Treatment AntibodyAntibody Group N^(a) Antibody Specificity Isotype 1 9 none NA^(b) NA(PBS alone) 2 10 Polyclonal Aβ1-42 mixed 3 0 mAb^(c) 2H3 Aβ1-12 IgG1 4 8mAb 10D5 Aβ1-16 IgG1 5 6 mAb 266 Aβ13-28 IgG1 6 8 mAb 21F12 Aβ33-42IgG2a Footnotes ^(a)Number of mice in group at termination of theexperiment. All groups started with 10 animals per group. ^(b)NA: notapplicable ^(c)mAb: monoclonal antibody

2. Materials and Methods

a. Preparation of the Antibodies

The anti-Aβ polyclonal antibody was prepared from blood collected fromtwo groups of animals. The first group consisted of 100 female SwissWebster mice, 6 to 8 weeks of age. They were immunized on days 0, 15,and 29 with 100 μg of AN1792 combined with CFA/IFA. A fourth injectionwas given on day 36 with one-half the dose of AN1792. Animals wereexsanguinated upon sacrifice at day 42, serum was prepared and the serawere pooled to create a total of 64 ml. The second group consisted of 24female mice isogenic with the PDAPP mice but nontransgenic for the humanAPP gene, 6 to 9 weeks of age. They were immunized on days 0, 14, 28 and56 with 100 μg of AN1792 combined with CFA/IFA. These animals were alsoexsanguinated upon sacrifice at day 63, serum was prepared and pooledfor a total of 14 ml. The two lots of sera were pooled. The antibodyfraction was purified using two sequential rounds of precipitation with50% saturated ammonium sulfate. The final precipitate was dialyzedagainst PBS and tested for endotoxin. The level of endotoxin was lessthan 1 EU/mg.

The anti-Aβ monoclonal antibodies were prepared from ascites fluid. Thefluid was first delipidated by the addition of concentrated sodiumdextran sulfate to ice-cold ascites fluid by stirring on ice to a reacha final concentration of 0.238%. Concentrated CaCl₂ was then added withstirring to reach a final concentration of 64 mM. This solution wascentrifuged at 10,000×g and the pellet was discarded. The supernatantwas stirred on ice with an equal volume of saturated ammonium sulfateadded dropwise. The solution was centrifuged again at 10,000×g and thesupernatant was discarded. The pellet was resuspended and dialyzedagainst 20 mM Tris-HCl, 0.4 M NaCl, pH 7.5. This fraction was applied toa Pharmacia FPLC Sepharose Q Column and eluted with a reverse gradientfrom 0.4 M to 0.275 M NaCl in 20 mM Tris-HCl, pH 7.5.

The antibody peak was identified by absorbance at 280 nm and appropriatefractions were pooled. The purified antibody preparation wascharacterized by measuring the protein concentration using the BCAmethod and the purity using SDS-PAGE. The pool was also tested forendotoxin. The level of endotoxin was less than 1 EU/mg. titers, titersless than 100 were arbitrarily assigned a titer value of 25.

3. Aβ and APP Levels in the Brain:

Following about six months of treatment with the various anti-Aβantibody preparations, brains were removed from the animals followingsaline perfusion. One hemisphere was prepared for immunohistochemicalanalysis and the second was used for the quantitation of Aβ and APPlevels. To measure the concentrations of various forms of beta amyloidpeptide and amyloid precursor protein (APP), the hemisphere wasdissected and homogenates of the hippocampal, cortical, and cerebellarregions were prepared in SM guanidine. These were serially diluted andthe level of amyloid peptide or APP was quantitated by comparison to aseries of dilutions of standards of Aβ peptide or APP of knownconcentrations in an ELISA format.

The levels of total Aβ and of Aβ1-42 measured by ELISA in homogenates ofthe cortex, and the hippocampus and the level of total Aβ in thecerebellum are shown in Tables 11, 12, and 13, respectively. The medianconcentration of total Aβ for the control group, inoculated with PBS,was 3.6-fold higher in the hippocampus than in the cortex (median of63,389 ng/g hippocampal tissue compared to 17,818 ng/g for the cortex).The median level in the cerebellum of the control group (30.6 ng/gtissue) was more than 2,000-fold lower than in the hippocampus. Theselevels are similar to those that we have previously reported forheterozygous PDAPP transgenic mice of this age (Johnson-Woods et al.,1997).

For the cortex, one treatment group had a median Aβ level, measured asAβ1-42, which differed significantly from that of the control group(p<0.05), those animals receiving the polyclonal anti-Aβ antibody asshown in Table 13. The median level of Aβ1-42 was reduced by 65%,compared to the control for this treatment group. The median levels ofAβ1-42 were also significantly reduced by 55% compared to the control inone additional treatment group, those animals dosed with the mAb 10D5(p=0.0433).

TABLE 13 CORTEX Medians Means Total Aβ Aβ42 Total Aβ Aβ42 TreatmentELISA ELISA ELISA ELISA Group N^(a) value^(b) P value^(c) % Change valueP value % Change value value PBS 9 17818 NA^(d) NA 13802 NA NA 16150 +/−7456^(e) 12621 +/− 5738  Polyclonal 10 6160 0.0055 −65 4892 0.0071 −655912 +/− 4492 4454 +/− 3347 anti-Aβ42 mAb 10D5 8 7915 0.1019 −56 62140.0433 −55 9695 +/− 6929 6943 +/− 3351 mAb 266 6 9144 0.1255 −49 84810.1255 −39 9204 +/− 9293 7489 +/− 6921 mAb 21F12 8 15158 0.2898 −1513578 0.7003  −2 12481 +/− 7082  11005 +/− 6324  Footnotes: ^(a)Numberof animals per group at the end of the experiment ^(b)ng/g tissue^(c)Mann Whitney analysis ^(d)NA: not applicable ^(e)Standard Deviation

In the hippocampus, the median percent reduction of total Aβ associatedwith treatment with polyclonal anti-Aβ antibody (50%, p=0.0055) was notas great as that observed in the cortex (65%) (Table 14). However, theabsolute magnitude of the reduction was almost 3-fold greater in thehippocampus than in the cortex, a net reduction of 31,683 ng/g tissue inthe hippocampus versus 11,658 ng/g tissue in the cortex. When measuredas the level of the more amyloidogenic form of Aβ, Aβ1-42, rather thanas total Aβ, the reduction achieved with the polyclonal antibody wassignificant (p=0.0025). The median levels in groups treated with themAbs 10D5 and 266 were reduced by 33% and 21%, respectively.

TABLE 14 HIPPOCAMPUS Medians Means Total Aβ Aβ42 Total Aβ Aβ42 TreatmentELISA P % ELISA P % ELISA ELISA Group N^(a) value^(b) value^(c) Changevalue value Change value value PBS 9 63389 NA^(d) NA 54429 NA NA 58351+/− 13308^(e) 52801 +/− 14701 Polyclonal 10 31706 0.0055 −50 271270.0025 −50 30058 +/− 22454 24853 +/− 18262 anti-Aβ42 mAb 10D5 8 467790.0675 −26 36290 0.0543 −33 44581 +/− 18632 36465 +/− 17146 mAb 266 648689 0.0990 −23 43034 0.0990 −21 36419 +/− 27304 32919 +/− 25372 mAb21F12 8 51563 0.7728 −19 47961 0.8099 −12 57327 +/− 28927 50305 +/−23927 Footnotes: ^(a)Number of animals per group at the end of theexperiment ^(b)ng/g tissue ^(c)Mann Whitney analysis ^(d)NA: notapplicable ^(e)Standard Deviation

Total Aβ was also measured in the cerebellum (Table 15). Those groupsdosed with the polyclonal anti-Aβ and the 266 antibody showedsignificant reductions of the levels of total Aβ (43% and 46%, p=0.0033and p=0.0184, respectively) and that group treated with 10D5 had a nearsignificant reduction (29%, p=0.0675).

TABLE 15 CEREBELLUM Medians Means Total Aβ Total Aβ Treatment ELISA P %ELISA Group N^(a) value^(b) value^(c) Change value PBS 9 30.64 NA^(d) NA40.00 +/− 31.89^(e) Polyclonal 10 17.61 0.0033 −43 18.15 +/− 4.36anti-Aβ42 mAb 10D5 8 21.68 0.0675 −29 27.29 +/− 19.43 mAb 266 6 16.590.0184 −46 19.59 +/− 6.59 mAb 21F12 8 29.80 >0.9999  −3 32.88 +/− 9.90Footnotes: ^(a)Number of animals per group at the end of the experiment^(b)ng/g tissue ^(c)Mann Whitney analysis ^(d)NA: not applicable^(e)Standard Deviation

APP concentration was also determined by ELISA in the cortex andcerebellum from antibody-treated and control, PBS-treated mice. Twodifferent APP assays were utilized. The first, designated APP-α/FL,recognizes both APP-alpha (α, the secreted form of APP which has beencleaved within the Aβ sequence), and full-length forms (FL) of APP,while the second recognizes only APP-α. In contrast to thetreatment-associated diminution of Aβ in a subset of treatment groups,the levels of APP were virtually unchanged in all of the treatedcompared to the control animals. These results indicate that theimmunizations with Aβ antibodies deplete Aβ without depleting APP.

In summary, Aβ levels were significantly reduced in the cortex,hippocampus and cerebellum in animals treated with the polyclonalantibody raised against AN1792. To a lesser extent monoclonal antibodiesto the amino terminal region of Aβ1-42, specifically amino acids 1-16and 13-28 also showed significant treatment effects.

4. Histochemical Analyses:

The morphology of Aβ-immunoreactive plaques in subsets of brains frommice in the PBS, polyclonal Aβ42, 21F12, 266 and 10D5 treatment groupswas qualitatively compared to that of previous studies in which standardimmunization procedures with Aβ42 were followed.

The largest alteration in both the extent and appearance of amyloidplaques occurred in the animals immunized with the polyclonal Aβ42antibody. The reduction of amyloid load, eroded plaque morphology andcell-associated Aβ immunoreactivity closely resembled effects producedby the standard immunization procedure. These observations support theELISA results in which significant reductions in both total Aβ3 and Aβ42were achieved by administration of the polyclonal Aβ42 antibody.

In similar qualitative evaluations, amyloid plaques in the 10D5 groupwere also reduced in number and appearance, with some evidence ofcell-associated Aβ immunoreactivity. Major differences were not seenwhen the 21F12 and 266 groups were compared with the PBS controls.

5. Measurement of Antibody Titers:

A subset of three randomly chosen mice from each group were bled justprior to each intraperitoneal inoculation, for a total of 30 bleeds.Antibody titers were measured as Aβ1-42-binding antibody using asandwich ELISA with plastic multi-well plates coated with Aβ1-42 asdescribed in detail in the General Materials and Methods. Mean titersfor each bleed are shown in FIGS. 16-18 for the polyclonal antibody andthe monoclonals 10D5 and 21F12, respectively. Titers averaged about1:1000 over this time period for the polyclonal antibody preparation andwere slightly above this level for the 10D5- and 21F12-treated animals.

6. Lymphoproliferative Responses

Aβ-dependent lymphoproliferation was measured using spleen cellsharvested eight days following the final antibody infusion. Freshlyharvested cells, 10⁵ per well, were cultured for 5 days in the presenceof Aβ1-40 at a concentration of 5 μM for stimulation. As a positivecontrol, additional cells were cultured with the T cell mitogen, PHA,and, as a negative control, cells were cultured without added peptide.

Splenocytes from aged PDAPP mice passively immunized with variousanti-Aβ antibodies were stimulated in vitro with AN1792 andproliferative and cytokine responses were measured. The purpose of theseassays was to determine if passive immunization facilitated antigenpresentation, and thus priming of T cell responses specific for AN1792.No AN1792-specific proliferative or cytokine responses were observed inmice passively immunized with the anti-Aβ antibodies.

B. Study 2

In a second study, treatment with antibody 10D5 was repeated and twoadditional anti-Aβ antibodies were tested, monoclonal antibodies 3D6(Aβ₁₋₅) and 16C11 (Aβ₃₃₋₄₂). Control groups received either PBS or anirrelevant isotype-matched antibody (TM2a). The mice were older (11.5-12month old heterozygotes) than in the previous study; otherwise theexperimental design was the same. Once again, after six months oftreatment, 10D5 reduced plaque burden by greater than 80% relative toeither the PBS or isotype-matched antibody controls (p=0.003). One ofthe other antibodies against Aβ, 3D6, was equally effective, producingan 86% reduction (p=0.003). In contrast, the third antibody against thepeptide, 16C11, failed to have any effect on plaque burden. Similarfindings were obtained with Aβ₄₂ ELISA measurements. These resultsdemonstrate that an antibody response against Aβ peptide, in the absenceof T cell immunity, is sufficient to decrease amyloid deposition inPDAPP mice, but that not all anti-Aβ antibodies are efficacious.Antibodies directed to epitopes comprising amino acids 1-5 or 3-7 of Aβare particularly efficacious.

These studies demonstrate that passively administered antibodies againstAβ reduced the extent of plaque deposition in a mouse model ofAlzheimer's disease. When held at modest serum concentrations (25-70μg/ml), the antibodies gained access to the CNS at levels sufficient todecorate β-amyloid plaques. Antibody entry into the CNS was not due toabnormal leakage of the blood-brain barrier since there was no increasein vascular permeability as measured by Evans Blue in PDAPP mice. Inaddition, the concentration of antibody in the brain parenchyma of agedPDAPP mice was the same as in non-transgenic mice, representing 0.1% ofthe antibody concentration in serum (regardless of isotype).

C. Study 3: Monitoring of Antibody Binding

To determine whether antibodies against Aβ could be acting directlywithin the CNS, brains taken from saline-perfused mice at the end of theExample XII, were examined for the presence of theperipherally-administered antibodies. Unfixed cryostat brain sectionswere exposed to a fluorescent reagent against mouse immunoglobulin (goatanti-mouse IgG-Cy3). Plaques within brains of the 10D5 and 3D6 groupswere strongly decorated with antibody, while there was no staining inthe 16C11 group. To reveal the full extent of plaque deposition, serialsections of each brain were first immunoreacted with an anti-Aβantibody, and then with the secondary reagent. 10D5 and 3D6, followingperipheral administration, gained access to most plaques within the CNS.The plaque burden was greatly reduced in these treatment groups comparedto the 16C11 group. These data indicate that peripherally administeredantibodies can enter the CNS where they can directly trigger amyloidclearance. It is likely that 16C11 also had access to the plaques butwas unable to bind to the plaques.

XII. Prevention and Treatment of Human Subjects

A single-dose phase I trial is performed to determine safety in humans.A therapeutic agent is administered in increasing dosages to differentpatients starting from about 0.01 the level of presumed efficacy, andincreasing by a factor of three until a level of about 10 times theeffective mouse dosage is reached.

A phase II trial is performed to determine therapeutic efficacy.Patients with early to mid Alzheimer's Disease defined using Alzheimer'sdisease and Related Disorders Association (ADRDA) criteria for probableAD are selected. Suitable patients score in the 12-26 range on theMini-Mental State Exam (MMSE). Other selection criteria are thatpatients are likely to survive the duration of the study and lackcomplicating issues such as use of concomitant medications that mayinterfere. Baseline evaluations of patient function are made usingclassic psychometric measures, such as the MMSE, and the ADAS, which isa comprehensive scale for evaluating patients with Alzheimer's Diseasestatus and function. These psychometric scales provide a measure ofprogression of the Alzheimer's condition. Suitable qualitative lifescales can also be used to monitor treatment. Disease progression canalso be monitored by MRI. Blood profiles of patients can also bemonitored including assays of immunogen-specific antibodies and T-cellsresponses.

Following baseline measures, patients begin receiving treatment. Theyare randomized and treated with either therapeutic agent or placebo in ablinded fashion. Patients are monitored at least every six months.Efficacy is determined by a significant reduction in progression of atreatment group relative to a placebo group.

A second phase II trial is performed to evaluate conversion of patientsfrom non-Alzheimer's Disease early memory loss, sometimes referred to asage-associated memory impairment (AAMI) or mild cognitive impairment(MCI), to probable Alzheimer's disease as defined as by ADRDA criteria.Patients with high risk for conversion to Alzheimer's Disease areselected from a non-clinical population by screening referencepopulations for early signs of memory loss or other difficultiesassociated with pre-Alzheimer's symptomatology, a family history ofAlzheimer's Disease, genetic risk factors, age, sex, and other featuresfound to predict high-risk for Alzheimer's Disease. Baseline scores onsuitable metrics including the MMSE and the ADAS together with othermetrics designed to evaluate a more normal population are collected.These patient populations are divided into suitable groups with placebocomparison against dosing alternatives with the agent. These patientpopulations are followed at intervals of about six months, and theendpoint for each patient is whether or not he or she converts toprobable Alzheimer's Disease as defined by ADRDA criteria at the end ofthe observation.

XIII. General Materials and Methods

1. Measurement of Antibody Titers

Mice were bled by making a small nick in the tail vein and collectingabout 200 μl of blood into a microfuge tube. Guinea pigs were bled byfirst shaving the back hock area and then using an 18 gauge needle tonick the metatarsal vein and collecting the blood into microfuge tubes.Blood was allowed to clot for one hr at room temperature (RT), vortexed,then centrifuged at 14,000×g for 10 min to separate the clot from theserum. Serum was then transferred to a clean microfuge tube and storedat 4° C. until titered.

Antibody titers were measured by ELISA. 96-well microtiter plates(Costar EIA plates) were coated with 100 μl of a solution containingeither 10 μg/ml either Aβ42 or SAPP or other antigens as noted in eachof the individual reports in Well Coating Buffer (0.1 M sodiumphosphate, pH 8.5, 0.1% sodium azide) and held overnight at RT. Thewells were aspirated and sera were added to the wells starting at a1/100 dilution in Specimen Diluent (0.014 M sodium phosphate, pH 7.4,0.15 M NaCl, 0.6% bovine serum albumin, 0.05% thimerosal). Seven serialdilutions of the samples were made directly in the plates in three-foldsteps to reach a final dilution of 1/218,700. The dilutions wereincubated in the coated-plate wells for one hr at RT. The plates werethen washed four times with PBS containing 0.05% Tween 20. The secondantibody, a goat anti-mouse Ig conjugated to horseradish peroxidase(obtained from Boehringer Mannheim), was added to the wells as 100 μl ofa 1/3000 dilution in Specimen Diluent and incubated for one hr at RT.Plates were again washed four times in PBS, Tween 20. To develop thechromogen, 100 μl of Slow TMB (3,3′,5,5′-tetramethyl benzidine obtainedfrom Pierce Chemicals) was added to each well and incubated for 15 minat RT. The reaction was stopped by the addition of 25 μl of 2 M H₂SO₄.The color intensity was then read on a Molecular Devices Vmax at (450nm-650 nm).

Titers were defined as the reciprocal of the dilution of serum givingone half the maximum OD. Maximal OD was generally taken from an initial1/100 dilution, except in cases with very high titers, in which case ahigher initial dilution was necessary to establish the maximal OD. Ifthe 50% point fell between two dilutions, a linear extrapolation wasmade to calculate the final titer. To calculate geometric mean antibodytiters, titers less than 100 were arbitrarily assigned a titer value of25.

2. Lymphocyte Proliferation Assay

Mice were anesthetized with isoflurane. Spleens were removed and rinsedtwice with 5 ml PBS containing 10% heat-inactivated fetal bovine serum(PBS-FBS) and then homogenized in a 50° Centricon unit (Dako A/S,Denmark) in 1.5 ml PBS-FBS for 10 sec at 100 rpm in a Medimachine (Dako)followed by filtration through a 100 micron pore size nylon mesh.Splenocytes were washed once with 15 ml PBS-FBS, then pelleted bycentrifugation at 200×g for 5 min. Red blood cells were lysed byresuspending the pellet in 5 mL buffer containing 0.15 M NH4Cl, 1 MKHCO3, 0.1 M NaEDTA, pH 7.4 for five min at RT. Leukocytes were thenwashed as above. Freshly isolated spleen cells (10⁵ cells per well) werecultured in triplicate sets in 96-well U-bottomed tissue culture-treatedmicrotiter plates (Corning, Cambridge, Mass.) in RPMI 1640 medium (JRHBiosciences, Lenexa, Kans.) supplemented with 2.05 mM L glutamine, 1%Penicillin/Streptomycin, and 10% heat-inactivated FBS, for 96 hr at 37°C. Various Aβ peptides, Aβ1-16, Aβ1-40, Aβ1-42 or Aβ40-1 reversesequence protein were also added at doses ranging from 5 to 0.18micromolar in four steps. Cells in control wells were cultured withConcanavalin A (Con A) (Sigma, cat. #C-5275, at 1 microgram/ml) withoutadded protein. Cells were pulsed for the final 24 hr with 3H-thymidine(1 μCi/well obtained from Amersham Corp., Arlington Heights Ill.). Cellswere then harvested onto UniFilter plates and counted in a Top CountMicroplate Scintillation Counter (Packard Instruments, Downers Grove,Ill.). Results are expressed as counts per minute (cpm) of radioactivityincorporated into insoluble macromolecules.

4. Brain Tissue Preparation

After euthanasia, the brains were removed and one hemisphere wasprepared for immunohistochemical analysis, while three brain regions(hippocampus, cortex and cerebellum) were dissected from the otherhemisphere and used to measure the concentration of various Aβ proteinsand APP forms using specific ELISAs (Johnson-Wood et al., supra).

Tissues destined for ELISAs were homogenized in 10 volumes of ice-coldguanidine buffer (5.0 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0). Thehomogenates were mixed by gentle agitation using an Adams Nutator(Fisher) for three to four hr at RT, then stored at −20° C. prior toquantitation of Aβ and APP. Previous experiments had shown that theanalytes were stable under this storage condition, and that synthetic Aβprotein (Bachem) could be quantitatively recovered when spiked intohomogenates of control brain tissue from mouse littermates (Johnson-Woodet al., supra).

5. Measurement of Aβ Levels

The brain homogenates were diluted 1:10 with ice cold Casein Diluent(0.25% casein, PBS, 0.05% sodium azide, 20 μg/ml aprotinin, 5 mM EDTA pH8.0, 10 μg/ml leupeptin) and then centrifuged at 16,000×g for 20 min at4° C. The synthetic Aβ protein standards (1-42 amino acids) and the APPstandards were prepared to include 0.5 M guanidine and 0.1% bovine serumalbumin (BSA) in the final composition. The “total” Aβ sandwich ELISAutilizes monoclonal antibody monoclonal antibody 266, specific for aminoacids 13-28 of Aβ (Seubert, et al.), as the capture antibody, andbiotinylated monoclonal antibody 3D6, specific for amino acids 1-5 of Aβ(Johnson-Wood, et al), as the reporter antibody. The 3D6 monoclonalantibody does not recognize secreted APP or full-length APP, but detectsonly Aβ species with an amino-terminal aspartic acid. This assay has alower limit of sensitivity of ˜50 ñg/ml (11 ñM) and shows nocross-reactivity to the endogenous murine Aβ protein at concentrationsup to 1 ng/ml (Johnson-Wood et al., supra).

The Aβ1-42 specific sandwich ELISA employs mAβ 21F12, specific for aminoacids 33-42 of Aβ (Johnson-Wood, et al.), as the capture antibody.Biotinylated mAβ 3D6 is also the reporter antibody in this assay whichhas a lower limit of sensitivity of about 125 μg/ml (28 μM, Johnson-Woodet al.). For the Aβ ELISAs, 100 μl of either mAβ 266 (at 10 μg/ml) ormAβ 21F12 at (5 μg/ml) was coated into the wells of 96-well immunoassayplates (Costar) by overnight incubation at RT. The solution was removedby aspiration and the wells were blocked by the addition of 200 μl of0.25% human serum albumin in PBS buffer for at least 1 hr at RT.Blocking solution was removed and the plates were stored desiccated at4° C. until used. The plates were rehydrated with Wash Buffer[Tris-buffered saline (0.15 M NaCl, 0.01 M Tris-HCl, pH 7.5), plus 0.05%Tween 20] prior to use. The samples and standards were added intriplicate aliquots of 100 μl per well and then incubated overnight at4° C. The plates were washed at least three times with Wash Bufferbetween each step of the assay. The biotinylated mAβ 3D6, diluted to 0.5μg/ml in Casein Assay Buffer (0.25% casein, PBS, 0.05% Tween 20, pH7.4), was added and incubated in the wells for 1 hr at RT. Anavidin-horseradish peroxidase conjugate, (Avidin-HRP obtained fromVector, Burlingame, Calif.), diluted 1:4000 in Casein Assay Buffer, wasadded to the wells for 1 hr at RT. The colorimetric substrate, SlowTMB-ELISA (Pierce), was added and allowed to react for 15 minutes at RT,after which the enzymatic reaction was stopped by the addition of 25 μl2 NH2SO4. The reaction product was quantified using a Molecular DevicesVmax measuring the difference in absorbance at 450 nm and 650 nm.

6. Measurement of APP Levels

Two different APP assays were utilized. The first, designated APP-α/FL,recognizes both APP-alpha (α) and full-length (FL) forms of APP. Thesecond assay is specific for APP-α. The APP-α/FL assay recognizessecreted APP including the first 12 amino acids of Aβ. Since thereporter antibody (2H3) is not specific to the α-clip-site, occurringbetween amino acids 612-613 of APP695 (Esch et al., Science 248,1122-1124 (1990)); this assay also recognizes full length APP (APP-FL).Preliminary experiments using immobilized APP antibodies to thecytoplasmic tail of APP-FL to deplete brain homogenates of APP-FLsuggest that approximately 30-40% of the APP-α/FL APP is FL (data notshown). The capture antibody for both the APP-α/FL and APP-α assays ismAb 8E5, raised against amino acids 444 to 592 of the APP695 form (Gameset al., supra). The reporter mAb for the APP-α/FL assay is mAb 2H3,specific for amino acids 597-608 of APP695 (Johnson-Wood et al., supra)and the reporter antibody for the APP-α assay is a biotinylatedderivative of mAb 16H9, raised to amino acids 605 to 611 of APP. Thelower limit of sensitivity of the APP-α/FL assay is about 11 ng/ml (150ρM) (Johnson-Wood et al.) and that of the APP-α specific assay is 22ng/ml (0.3 nM). For both APP assays, mAb 8E5 was coated onto the wellsof 96-well EIA plates as described above for mAb 266. Purified,recombinant secreted APP-α was used as the reference standard for theAPP-α assay and the APP-α/FL assay (Esch et al., supra). The brainhomogenate samples in 5 M guanidine were diluted 1:10 in ELISA SpecimenDiluent (0.014 M phosphate buffer, pH 7.4, 0.6% bovine serum albumin,0.05% thimerosal, 0.5 M NaCl, 0.1% NP40). They were then diluted 1:4 inSpecimen Diluent containing 0.5 M guanidine. Diluted homogenates werethen centrifuged at 16,000×g for 15 seconds at RT. The APP standards andsamples were added to the plate in duplicate aliquots and incubated for1.5 hr at RT. The biotinylated reporter antibody 2H3 or 16H9 wasincubated with samples for 1 hr at RT. Streptavidin-alkaline phosphatase(Boehringer Mannheim), diluted 1:1000 in specimen diluent, was incubatedin the wells for 1 hr at RT. The fluorescent substrate4-methyl-umbellipheryl-phosphate was added for a 30-min RT incubationand the plates were read on a Cytofluor™ 2350 fluorimeter (Millipore) at365 nm excitation and 450 nm emission.

7. Immunohistochemistry

Brains were fixed for three days at 40 C in 4% paraformaldehyde in PBSand then stored from one to seven days at 4° C. in 1% paraformaldehyde,PBS until sectioned. Forty-micron-thick coronal sections were cut on avibratome at RT and stored in cryoprotectant (30% glycerol, 30% ethyleneglycol in phosphate buffer) at −20° C. prior to immunohistochemicalprocessing. For each brain, six sections at the level of the dorsalhippocampus, each separated by consecutive 240 μm intervals, wereincubated overnight with one of the following antibodies: (1) abiotinylated anti-Aβ (mAb, 3D6, specific for human Aβ) diluted to aconcentration of 2 μg/ml in PBS and 1% horse serum; or (2) abiotinylated mAb specific for human APP, 8E5, diluted to a concentrationof 3 μg/ml in PBS and 1.0% horse serum; or (3) a mAb specific for glialfibrillary acidic protein (GFAP; Sigma Chemical Co.) diluted 1:500 with0.25% Triton X-100 and 1% horse serum, in Tris-buffered saline, pH 7.4(TBS); or (4) a mAb specific for CD11b, MAC-1 antigen, (ChemiconInternational) diluted 1:100 with 0.25% Triton X-100 and 1% rabbit serumin TBS; or (5) a mAb specific for MHC II antigen, (Pharmingen) diluted1:100 with 0.25% Triton X-100 and 1% rabbit serum in TBS; or (6) a ratmAb specific for CD 43 (Pharmingen) diluted 1:100 with 1% rabbit serumin PBS or (7) a rat mAb specific for CD 45RA (Pharmingen) diluted 1:100with 1% rabbit serum in PBS; or (8) a rat monoclonal Aβ specific for CD45RB (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS; or (9) arat monoclonal Aβ specific for CD 45 (Pharmingen) diluted 1:100 with 1%rabbit serum in PBS; or (10) a biotinylated polyclonal hamster Aβspecific for CD3e (Pharmingen) diluted 1:100 with 1% rabbit serum in PBSor (11) a rat mAb specific for CD3 (Serotec) diluted 1:200 with 1%rabbit serum in PBS; or with (12) a solution of PBS lacking a primaryantibody containing 1% normal horse serum.

Sections reacted with antibody solutions listed in 1, 2 and 6-12 abovewere pretreated with 1.0% Triton X-100, 0.4% hydrogen peroxide in PBSfor 20 min at RT to block endogenous peroxidase. They were nextincubated overnight at 4° C. with primary antibody. Sections reactedwith 3D6 or 8E5 or CD3e mAbs were then reacted for one hr at RT with ahorseradish peroxidase-avidin-biotin-complex with kit components “A” and“B” diluted 1:75 in PBS (Vector Elite Standard Kit, Vector Labs,Burlingame, Calif.). Sections reacted with antibodies specific for CD45RA, CD 45RB, CD 45, CD3 and the PBS solution devoid of primaryantibody were incubated for 1 hour at RT with biotinylated anti-rat IgG(Vector) diluted 1:75 in PBS or biotinylated anti-mouse IgG (Vector)diluted 1:75 in PBS, respectively. Sections were then reacted for one hrat RT with a horseradish peroxidase-avidin-biotin-complex with kitcomponents “A” and “B” diluted 1:75 in PBS (Vector Elite Standard Kit,Vector Labs, Burlingame, Calif.).

Sections were developed in 0.01% hydrogen peroxide, 0.05%3,3′-diaminobenzidine (DAB) at RT. Sections destined for incubation withthe GFAP-, MAC-1- AND MHC II-specific antibodies were pretreated with0.6% hydrogen peroxide at RT to block endogenous peroxidase thenincubated overnight with the primary antibody at 4° C. Sections reactedwith the GFAP antibody were incubated for 1 hr at RT with biotinylatedanti-mouse IgG made in horse (Vector Laboratories; Vectastain Elite ABCKit) diluted 1:200 with TBS. The sections were next reacted for one hrwith an avidin-biotin-peroxidase complex (Vector Laboratories;Vectastain Elite ABC Kit) diluted 1:1000 with TBS. Sections incubatedwith the MAC-1- or MHC II-specific monoclonal antibody as the primaryantibody were subsequently reacted for 1 hr at RT with biotinylatedanti-rat IgG made in rabbit diluted 1:200 with TBS, followed byincubation for one hr with avidin-biotin-peroxidase complex diluted1:1000 with TBS. Sections incubated with GFAP-, MAC-1- and MHCII-specific antibodies were then visualized by treatment at RT with0.05% DAB, 0.01% hydrogen peroxide, 0.04% nickel chloride, TBS for 4 and11 min, respectively.

Immunolabeled sections were mounted on glass slides (VWR, Superfrostslides), air dried overnight, dipped in Propar (Anatech) and overlaidwith coverslips using Permount (Fisher) as the mounting medium.

To counterstain Aβ plaques, a subset of the GFAP-positive sections weremounted on Superfrost slides and incubated in aqueous 1% Thioflavin S(Sigma) for 7 min following immunohistochemical processing. Sectionswere then dehydrated and cleared in Propar, then overlaid withcoverslips mounted with Permount.

8. Image Analysis

A Videometric 150 Image Analysis System (Oncor, Inc., Gaithersburg, Md.)linked to a Nikon Microphot-FX microscope through a CCD video camera anda Sony Trinitron monitor was used for quantification of theimmunoreactive slides. The image of the section was stored in a videobuffer and a color- and saturation-based threshold was determined toselect and calculate the total pixel area occupied by the immunolabeledstructures. For each section, the hippocampus was manually outlined andthe total pixel area occupied by the hippocampus was calculated. Thepercent amyloid burden was measured as: (the fraction of the hippocampalarea containing Aβ deposits immunoreactive with mAb 3D6)×100. Similarly,the percent neuritic burden was measured as: (the fraction of thehippocampal area containing dystrophic neurites reactive with monoclonalantibody 8E5)×100. The C-Imaging System (Compix, Inc., CranberryTownship, Pa.) operating the Simple 32 Software Application program waslinked to a Nikon Microphot-FX microscope through an Optronics cameraand used to quantitate the percentage of the retrospenial cortexoccupied by GFAP-positive astrocytes and MAC-1- and MHC II-positivemicroglia. The image of the immunoreacted section was stored in a videobuffer and a monochrome-based threshold was determined to select andcalculate the total pixel area occupied by immunolabeled cells. For eachsection, the retrosplenial cortex (RSC) was manually outlined and thetotal pixel area occupied by the RSC was calculated. The percentastrocytosis was defined as: (the fraction of RSC occupied byGFAP-reactive astrocytes)×100. Similarly, percent microgliosis wasdefined as: (the fraction of the RSC occupied by MAC-1- or MHCII-reactive microglia)×100. For all image analyses, six sections at thelevel of the dorsal hippocampus, each separated by consecutive 240 μmintervals, were quantitated for each animal. In all cases, the treatmentstatus of the animals was unknown to the observer.

XIV: Ex Vivo Screening Assay for Activity of an Antibody Against AmyloidDeposits

An ex vivo assay in which primary microglial cells were cultured withunfixed cryostat sections of either PDAPP mouse or human AD brains wasestablished, in order to examine the effects of antibodies on plaqueclearance. Microglial cells were obtained from the cerebral cortices ofneonate DBA/2N mice (1-3 days). The cortices were mechanicallydissociated in HBSS⁻ (Hanks' Balanced Salt Solution, Sigma) with 50μg/ml DNase I (Sigma). The dissociated cells were filtered with a 100 μmcell strainer (Falcon), and centrifuged at 1000 rpm for 5 minutes. Thepellet was resuspended in growth medium (high glucose DMEM, 10% FBS, 25ng/ml rmGM-CSF), and the cells were plated at a density of 2 brains perT-75 plastic culture flask. After 7-9 days, the flasks were rotated onan orbital shaker at 200 rpm for 2 h at 37° C. The cell suspension wascentrifuged at 1000 rpm and resuspended in the assay medium.

10-μm cryostat sections of PDAPP mouse or human AD brains (post-morteminterval<3 hr) were thaw mounted onto poly-lysine coated round glasscoverslips and placed in wells of 24-well tissue culture plates. Thecoverslips were washed twice with assay medium consisting of H-SFM(Hybridoma-serum free medium, Gibco BRL) with 1% FBS, glutamine,penicillin/streptomycin, and 5 ng/ml rmGM-CSF (R&D). Control or anti-Aβantibodies were added at a 2× concentration (5 μg/ml final) for 1 hour.The microglial cells were then seeded at a density of 0.5×10⁶ cells/mlassay medium. The cultures were maintained in a humidified incubator(37° C., 5% CO₂) for 24 hr or more. At the end of the incubation, thecultures were fixed with 4% paraformaldehyde and permeabilized with 0.1%Triton-X100. The sections were stained with biotinylated 3D6 followed bya streptavidin/Cy3 conjugate (Jackson ImmunoResearch). The exogenousmicroglial cells were visualized by a nuclear stain (DAPI). The cultureswere observed with an inverted fluorescent microscope (Nikon, TE300) andphotomicrographs were taken with a SPOT digital camera using SPOTsoftware (Diagnostic instruments). For Western blot analysis, thecultures were extracted in 8M urea, diluted 1:1 in reducing tricinesample buffer and loaded onto a 16% tricine gel (Novex). After transferonto immobilon, blots were exposed to 5 μg/ml of the pabAβ342 followedby an HRP-conjugated anti-mouse antibody, and developed with ECL(Amersham)

When the assay was performed with PDAPP brain sections in the presenceof antibody 16C11 (one of the antibodies against Aβ that was notefficacious in vivo), β-amyloid plaques remained intact and nophagocytosis was observed. In contrast, when adjacent sections werecultured in the presence of 10D5, the amyloid deposits were largely goneand the microglial cells showed numerous phagocytic vesicles containingAβ. Identical results were obtained with AD brain sections; 10D5 inducedphagocytosis of AD plaques, while 16C11 was ineffective. In addition,the assay provided comparable results when performed with either mouseor human microglial cells, and with mouse, rabbit, or primate antibodiesagainst Aβ.

Table 16 shows results obtained with several antibodies against Aβ,comparing their abilities to induce phagocytosis in the ex vivo assayand to reduce in vivo plaque burden in passive transfer studies.Although 16C11 and 21F12 bound to aggregated synthetic Aβ peptide withhigh avidity, these antibodies were unable to react with β-amyloidplaques in unfixed brain sections, could not trigger phagocytosis in theex vivo assay, and were not efficacious in vivo. 10D5, 3D6, and thepolyclonal antibody against Aβ were active by all three measures. The22C8 antibody binds more strongly to an analog form of natural Aβ inwhich aspartic acid at positions 1 and 7 is replaced with iso-asparticacid. These results show that efficacy in vivo is due to direct antibodymediated clearance of the plaques within the CNS, and that the ex vivoassay is predictive of in vivo efficacy.

The same assay has been used to test clearing of an antibody against afragment of synuclein referred to as NAC. Synuclein has been shown to bean amyloid plaque-associated protein. An antibody to NAC was contactedwith a brain tissue sample containing amyloid plaques, an microglialcells, as before. Rabbit serum was used as a control. Subsequentmonitoring showed a marked reduction in the number and size of plaquesindicative of clearing activity of the antibody.

TABLE 16 The ex vivo assay as predictor of in vivo efficacy. Avidity forBinding to aggregated β-amyloid Ex vivo In vivo Antibody Isotype Aβ (pM)plaques efficacy efficacy monoclonal 3D6 IgG2b 470 + + + 10D5 IgG143 + + + 16C11 IgG1 90 − − − 21F12 IgG2a 500 − − − TM2a IgG1 — − − −polyclonal 1-42 mix 600 + + +

Confocal microscopy was used to confirm that Aβ was internalized duringthe course of the ex vivo assay. In the presence of control antibodies,the exogenous microglial cells remained in a confocal plane above thetissue, there were no phagocytic vesicles containing Aβ, and the plaquesremained intact within the section. In the presence of 10D5, nearly allplaque material was contained in vesicles within the exogenousmicroglial cells. To determine the fate of the internalized peptide,10D5 treated cultures were extracted with 8M urea at varioustime-points, and examined by Western blot analysis. At the one hour timepoint, when no phagocytosis had yet occurred, reaction with a polyclonalantibody against Aβ revealed a strong 4 kD band (corresponding to the Aβpeptide). Aβ immunoreactivity decreased at day 1 and was absent by day3. Thus, antibody-mediated phagocytosis of Aβ leads to its degradation.

To determine if phagocytosis in the ex vivo assay was Fc-mediated,F(ab′)2 fragments of the anti-Aβ antibody 3D6 were prepared. Althoughthe F(ab′)2 fragments retained their full ability to react with plaques,they were unable to trigger phagocytosis by microglial cells. Inaddition, phagocytosis with the whole antibody could be blocked by areagent against murine Fc receptors (anti-CD16/32). These data indicatethat in vivo clearance of Aβ occurs through Fc-receptor mediatedphagocytosis.

XV: Passage of Antibodies Through the Blood-Brain Barrier

This experiments described herein were performed in order to provideinformation on ability of antibodies to pass into the brain followingintravenous injection and to provide means for measuring theconcentration of antibody delivered to the brain following intravenousinjection into a peripheral tissue of either normal or PDAPP mice. Suchmeasurements are useful in predicting and determining effective dosages.

PDAPP or control normal mice were perfused with 0.9% NaCl. Brain regions(hippocampus or cortex) were dissected and rapidly frozen. Brain werehomogenized in 0.1% triton+protease inhibitors. Immunoglobulin wasdetected in the extracts by ELISA. Fab′2 Goat Anti-mouse IgG were coatedonto an RIA plate as capture reagent. The serum or the brain extractswere incubated for 1 hr. The isotypes were detected with anti-mouseIgG1-HRP or IgG2a-HRP or IgG2b-HRP (Caltag). Antibodies, regardless ofisotype, were present in the CNS at a concentration that is 1:1000 thatfound in the blood. For example, when the concentration of IgG1 wasthree times that of IgG2a in the blood, it was three times IgG2a in thebrain as well, both being present at 0.1% of their respective levels inthe blood. This result was observed in both transgenic and nontransgenicmice—so the PDAPP does not have a uniquely leaky blood brain barrier.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

1-57. (canceled)
 58. A method of therapeutically treating amyloid Aamyloidosis in a patient comprising administering to the patient amyloidA protein or serum amyloid A precursor protein, or a fragment thereof,which effects an immune response comprising antibodies against amyloid Aprotein, and an adjuvant that augments the immune response, to therebytreat amyloid A amyloidosis.
 59. The method of claim 58, wherein theamyloid A amyloidosis is associated with a preexisting or coexistinginflammatory disease or condition, an infection, a neoplasm, or alymphoproliferative disorder.
 60. The method of claim 58, wherein theamyloid A amyloidosis is associated with a heredofamilial syndrome. 61.The method of claim 58, wherein the amyloid A protein or serum amyloid Aprecursor protein, or a fragment thereof, is linked to a carriermolecule.
 62. The method of claim 58, wherein the adjuvant is selectedfrom the group consisting of QS21, monophosphoryl lipid, and alum. 63.The method of claim 58, wherein the immune response is characterized bya serum titer of the antibodies of at least 1:1000 with respect toamyloid A protein.
 64. The method of claim 63, wherein the immuneresponse is characterized by a serum titer of the antibodies of at least1:5000 with respect to amyloid A protein.
 65. The method of claim 58,wherein the immune response is characterized by a serum titer of theantibodies against amyloid A protein that is greater than about fourtimes higher than a serum titer of antibodies measured in apre-treatment control serum sample.
 66. The method of claim 58, wherebydeposition of amyloid A protein fibril aggregates is inhibited.
 67. Themethod of claim 58, whereby amyloid A protein fibril aggregates arecleared.
 68. A method of reducing risk or delaying onset of amyloid Aamyloidosis in a patient at risk of amyloid A amyloidosis comprisingadministering to the patient amyloid A protein or serum amyloid Aprecursor protein, or a fragment thereof, which effects an immuneresponse comprising antibodies against amyloid A protein, and anadjuvant that augments the immune response, to thereby effectprophylaxis of amyloid A amyloidosis.
 69. The method of claim 68,wherein the patient has or is at risk for an inflammatory disease orcondition, an infection, a neoplasm, or a lymphoproliferative disorder.70. The method of claim 68, wherein the patient has or is at risk for aheredofamilial syndrome.
 71. The method of claim 68, wherein the amyloidA protein or serum amyloid A precursor protein, or a fragment thereof,is linked to a carrier molecule.
 72. The method of claim 68, wherein theadjuvant is selected from the group consisting of QS21, monophosphoryllipid, and alum.
 73. The method of claim 68, wherein the immune responseis characterized by a serum titer of the antibodies of at least 1:1000with respect to the amyloid A protein.
 74. The method of claim 73,wherein the immune response is characterized by a serum titer of theantibodies of at least 1:5000 with respect to the amyloid A protein. 75.The method of claim 68, wherein the immune response is characterized bya serum titer of the antibodies against the amyloid A protein that isgreater than about four times higher than a serum titer of antibodiesmeasured in a pre-treatment control serum sample.
 76. The method ofclaim 68, whereby deposition of amyloid A protein fibril aggregates isinhibited.
 77. The method of claim 68, whereby amyloid A protein fibrilaggregates are cleared.
 78. A method of therapeutically treating apatient having AL amyloidosis characterized by the presence of amyloidlight chain-type protein fibrils comprising administering to the patientamyloid light chain-type protein or immunoglobulin light chain precursorprotein, or a fragment thereof, which effects an immune responsecomprising antibodies against amyloid light chain-type protein, and anadjuvant that augments the immune response, to thereby treat ALamyloidosis.
 79. The method of claim 78, wherein the AL amyloidosis isassociated with a preexisting or coexisting inflammatory disease orcondition or a lymphoproliferative disorder.
 80. The method of claim 79,wherein the lymphoproliferative disorder is a dyscrasia of the Blymphocyte lineage.
 81. The method of claim 78, wherein the amyloidlight chain-type protein or immunoglobulin light chain precursorprotein, or a fragment thereof, is linked to a carrier molecule.
 82. Themethod of claim 78, wherein the adjuvant is selected from the groupconsisting of QS21, monophosphoryl lipid, and alum.
 83. The method ofclaim 78, wherein the immune response is characterized by a serum titerof the antibodies of at least 1: 1000 with respect to amyloid lightchain-type protein.
 84. The method of claim 83, wherein the immuneresponse is characterized by a serum titer of the antibodies of at least1:5000 with respect to amyloid light chain-type protein.
 85. The methodof claim 78, wherein the immune response is characterized by a serumtiter of the antibodies against amyloid light chain-type protein that isgreater than about four times higher than a serum titer of antibodiesmeasured in a pre-treatment control serum sample.
 86. The method ofclaim 78, whereby deposition of amyloid light chain-type protein fibrilaggregates is inhibited.
 87. The method of claim 78, whereby amyloidlight chain-type protein fibril aggregates are cleared.
 88. A method ofreducing risk or delaying onset of AL amyloidosis in a patient at riskof AL amyloidosis comprising administering to the patient amyloid lightchain-type protein or immunoglobulin light chain precursor protein, or afragment thereof, which effects an immune response comprising antibodiesagainst amyloid light chain-type protein, and an adjuvant that augmentsthe immune response, to thereby effect prophylaxis of AL amyloidosis.89. The method of claim 88, wherein the patient has or is at risk for apreexisting or coexisting inflammatory disease or condition or alymphoproliferative disorder.
 90. The method of claim 89, wherein thelymphoproliferative disorder is a dyscrasia of the B lymphocyte lineage.91. The method of claim 88, wherein the amyloid light chain-type proteinor immunoglobulin light chain precursor protein, or a fragment thereof,is linked to a carrier molecule.
 92. The method of claim 88, wherein theadjuvant is selected from the group consisting of QS21, monophosphoryllipid, and alum.
 93. The method of claim 88, wherein the immune responseis characterized by a serum titer of the antibodies of at least 1:1000with respect to the amyloid light chain-type protein.
 94. The method ofclaim 93, wherein the immune response is characterized by a serum titerof the antibodies of at least 1:5000 with respect to the amyloid lightchain-type protein.
 95. The method of claim 88, wherein the immuneresponse is characterized by a serum titer of the antibodies against theamyloid light chain-type protein that is greater than about four timeshigher than a serum titer of antibodies measured in a pre-treatmentcontrol serum sample.
 96. The method of claim 88, whereby deposition ofamyloid light chain-type protein fibril aggregates is inhibited.
 97. Themethod of claim 88, whereby amyloid light chain-type protein fibrilaggregates are cleared.